A probiotic agent for repairing damage to a genital tract mucosal barrier, and a preparation method and application thereof
By combining probiotic strains WP502, LC97, and Lp18 with prebiotics, the problem of poor inhibition of Gardnerella vaginalis and repair of the reproductive tract mucosal barrier in existing technologies has been solved, achieving effective treatment of bacterial vaginosis and repair of the mucosal barrier.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU WECARE BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-06-23
- Publication Date
- 2026-07-24
Smart Images
Figure CN122445504A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology and relates to a probiotic agent for repairing damage to the reproductive tract mucosal barrier, its preparation method, and its application. Background Technology
[0002] Bacterial vaginosis is the most common vaginal infection in women, characterized by a typical disruption of the vaginal flora, primarily manifested as a sharp decline in lactobacilli-dominated flora. Clinically, it presents as thin vaginal discharge with a foul or fishy odor. The main pathogens causing vaginitis include Gardnerella vaginalis, Atobacillus aureus, and Prevotella vaginalis, with Gardnerella vaginalis being the core pathogen.
[0003] When the vaginal pH is higher than 4.5, Gardnerella vaginalis overgrows, producing more amines, which further increases the vaginal pH and exacerbates symptoms. Besides attaching to vaginal epithelial cells and releasing cytotoxins (vaginal hemolysin and sialidase) to disrupt the vaginal mucosal barrier, overgrowth of Gardnerella vaginalis also lowers the redox sites in the vaginal microbiome, leading to fewer lactobacilli, an increase in vaginitis-related pathogens, and vaginal microecological dysbiosis.
[0004] Currently, the treatment of bacterial vaginosis mainly involves two common methods: antibacterial drugs and microbial intervention. Antibacterial drugs usually employ antibiotics, but these have significant side effects and are prone to recurrence. Microbial intervention typically uses probiotics, which are microorganisms that can improve the health of the host when ingested in sufficient quantities. Common dominant probiotics (lactobacters) include *Lactobacillus curvatureii*, *Lactobacillus geranioli*, *Lactobacillus rhamnosus*, and *Lactobacillus jansii*. Lactobacillus protects vaginal health through multiple mechanisms: (1) Acid production: Lactobacillus can use glycogen in the vagina to produce lactic acid, maintaining a low pH environment in the vagina (usually between 3.8 and 4.5); this low pH environment is not conducive to the growth of most pathogenic bacteria, thus effectively preventing the invasion and reproduction of pathogenic bacteria; (2) Production of antibacterial substances: Lactobacillus can also produce antibacterial substances such as hydrogen peroxide and bacteriocins, which can directly inhibit or kill pathogenic bacteria, further enhancing the defense capabilities of the vaginal barrier; (3) Competitive exclusion: Lactobacillus competitively adsorbs epithelial cells, preventing the adhesion and colonization of other microorganisms, thereby reducing the chance of pathogen infection; this competitive exclusion mechanism effectively prevents the vaginal mucosal barrier function from being damaged, while also helping to maintain the stability of the vaginal microbiota; (4) Immune regulation: Lactobacillus metabolites can effectively reduce the secretion of pro-inflammatory factors, while also effectively stimulating the vaginal mucosal barrier to secrete anti-inflammatory factors, repairing the damaged vaginal mucosal barrier.
[0005] Clinically, the occurrence, development, and outcome of many diseases are related to damage to the reproductive tract mucosal barrier. Therefore, screening probiotics that can inhibit Gardnerella vaginalis and effectively repair damage to the reproductive tract mucosal barrier is of great value. Summary of the Invention
[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a probiotic agent for repairing damage to the reproductive tract mucosal barrier, its preparation method and application.
[0007] To achieve this objective, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides a probiotic agent for repairing damage to the mucosal barrier of the reproductive tract, wherein the strains in the probiotic agent include Lactobacillus rhamnosus WP502 strain with accession number CGMCC No.37035, Lactobacillus paracasei LC97 strain with accession number CGMCC No.32421, and Lactobacillus plantarum Lp18 strain with accession number CCTCC NO: M 20231524.
[0009] The Lactobacillus rhamnosus WP502 described in this invention has a broad-spectrum antibacterial effect against common vaginal pathogens. In particular, it has a very good antibacterial effect against Gardnerella vaginalis, the core pathogen of bacterial vaginosis. It can effectively prevent pathogens from attaching and multiplying, exert a competitive exclusion effect, inhibit their growth and toxicity, promote their expulsion, reduce the concentration of pathogens in the vagina, reduce the risk of infection, and has a strong potential to adhere to vaginal epithelial cells, thereby protecting the vaginal mucosal barrier from pathogenic microorganisms and exerting its probiotic function.
[0010] This invention creatively discovers that *Lactobacillus rhamnosus* WP502 strain, *Lactobacillus paracasei* LC97 strain, and *Lactobacillus plantarum* Lp18 strain have a certain synergistic effect in repairing damage to the reproductive tract mucosal barrier; the absence of any one of them reduces the repair effect. Furthermore, the bacterial agent described in this invention achieves its effect of repairing reproductive tract mucosal barrier damage based on host immune regulation.
[0011] Preferably, the ratio of viable counts of Lacticaseibacillus rhamnosus WP502, Lacticaseibacillus paracasei LC97, and Lactiplantibacillus plantarum Lp18 is (1-4):(1-2):(1-2).
[0012] The specific point values in (1-4) can all be selected from 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, etc., and the specific point values in (1-2) can all be selected from 1, 1.2, 1.5, 1.8, 2, etc. Other specific point values within the above range can also be selected, which will not be elaborated here.
[0013] Preferably, the probiotic agent further includes prebiotics.
[0014] Preferably, the probiotic agent further includes a protectant.
[0015] Preferably, the protective agent comprises any one or a combination of at least two of the following: sodium vitamin C, trehalose, soluble starch, or skim milk powder.
[0016] Preferably, the prebiotic content in the probiotic agent is 1-10% by mass, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Other specific values within the above range can be selected, and will not be elaborated here.
[0017] Preferably, the prebiotics include D-alloxanose, fructooligosaccharides, and inulin.
[0018] Preferably, the mass ratio of D-allulose, fructooligosaccharide and inulin is (1-3):(1-3):(1-3).
[0019] The specific point values in (1-3) can all be selected from 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values within the above range can also be selected, which will not be elaborated here.
[0020] In a second aspect, the present invention provides a method for preparing a probiotic agent for repairing damage to the reproductive tract mucosal barrier according to the first aspect. The preparation method includes: inoculating the bacterial strains into a culture medium, culturing, centrifuging to collect the bacterial sludge, mixing the bacterial sludge with a protective agent, and freeze-drying to obtain the product.
[0021] Preferably, after freeze-drying, the product is further mixed with prebiotics.
[0022] Thirdly, the present invention provides the application of the probiotic agent for repairing damage to the reproductive tract mucosal barrier as described in the first aspect in the preparation of a medicine having the effect of repairing damage to the reproductive tract mucosal barrier.
[0023] Fourthly, the present invention provides the use of the probiotic agent according to the first aspect for repairing damage to the reproductive tract mucosal barrier in the preparation of a medicine having the effect of treating bacterial vaginosis.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] The *Lactobacillus rhamnosus* WP502 strain described in this invention exhibits broad-spectrum antibacterial activity against common vaginal pathogens. Specifically, it demonstrates excellent antibacterial efficacy against *Gardnerella vaginalis*, the core pathogen of bacterial vaginosis. It effectively prevents pathogen attachment and reproduction, exerts competitive exclusion, inhibits its growth and toxicity, promotes its expulsion, reduces the concentration of pathogens in the vagina, and decreases the risk of infection. It also possesses a strong potential to adhere to vaginal epithelial cells, thereby protecting the vaginal mucosal barrier from pathogenic microorganisms and exerting its probiotic function. Combining *Lactobacillus rhamnosus* WP502, *Lactobacillus paracasei* LC97, and *Lactobacillus plantarum* Lp18 results in a superior therapeutic effect against bacterial vaginosis. Furthermore, the three strains exhibit a certain synergistic effect in achieving the aforementioned effects, and the addition of prebiotics can further enhance these effects. Attached Figure Description
[0026] Figure 1 This is a graph showing the self-cohesion results of Lactobacillus rhamnosus WP502.
[0027] Figure 2 This is a graph showing the copolymerization results of Lactobacillus rhamnosus WP502 and Gv.
[0028] Figure 3 This is a diagram showing the surface hydrophobicity of Lactobacillus rhamnosus WP502.
[0029] Figure 4 These are images of the vaginal opening and vaginal tissue of mice in each group.
[0030] Figure 5 This is the qPCR standard curve for Gv.
[0031] Figure 6 This is a graph showing the Gv load analysis of vaginal irrigation fluid in each group of mice.
[0032] Figure 7 These are pathological sections of vaginal tissue from each group of mice.
[0033] Figure 8This is a graph showing the average level analysis of vaginal inflammatory factors in each group of mice, where A represents IL-6, B represents IL-10, C represents TNF-α, D represents IL-17, E represents IL-1β, F represents MPO, and G represents SIgA.
[0034] Figure 9 This is a graph showing the content analysis of vaginal mucosal barrier protein factors in each group of mice, where A represents ZO-1, B represents GLDN1, and C represents OCLN.
[0035] Figure 10 This is a graph showing the β-diversity analysis of each group of mice.
[0036] Figure 11 This is a taxonomic analysis diagram of the vaginal flora at the phylum level in each group of mice.
[0037] Figure 12 This is a taxonomic analysis diagram of the vaginal flora at the genus level in each group of mice.
[0038] Figure 13 This is a statistical chart of species taxonomy analysis of the vaginal flora of mice in each group, where A represents Shigella, B represents Alcaligenes, C represents Enterococcus, and D represents Streptococcus. Detailed Implementation
[0039] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.
[0040] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):
[0041] The Candida albicans ATCC10231, Gardnerella vaginalis ATCC14018, Escherichia coli ATCC25922, Staphylococcus aureus ATCC6538, Salmonella ATCC14028, and Atobacillus vaginalis ATCC BAA-55 mentioned in the following examples were all purchased from Guangdong Microbial Culture Collection Center.
[0042] The Lactobacillus rhamnosus strain WP502 mentioned below is classified as Lactobacillus rhamnosus, with accession number CGMCC No. 37035, accession date of December 11, 2025, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0043] The Lactobacillus paracasei strain LC97 mentioned below is classified as Lactobacillus paracasei, with accession number CGMCC No. 32421, accession date of October 30, 2024, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0044] The Lp18 strain mentioned below is classified as Lactiplantibacillus plantarum Lp18, with accession number CCTCC NO: M 20231524, accession date August 23, 2023, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.
[0045] The BHI culture medium used in the following examples was purchased from Haibo Biotechnology Co., Ltd.; defibrinated sheep blood was purchased from Yuanye Biotechnology Co., Ltd.; and the TNF-α, IL-6, IL-10, IL-1β, SIgA, MPO, and IL-17 ELISA kits were purchased from Wuhan Beiyinlai Biotechnology Co., Ltd.
[0046] The culture medium formulations mentioned below are as follows:
[0047] MRS medium: glucose 20 g / L, yeast peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, anhydrous sodium acetate 5 g / L, Tween-80 1 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.58 g / L, manganese sulfate monohydrate 0.19 g / L, and water, pH 6.8; 1.5% agar powder is added to the solid medium.
[0048] YPD medium: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, water, pH 7.0; 1.5% agar powder is added to the solid medium.
[0049] LB medium: 10 g / L tryptone, 5 g / L yeast, 5 g / L NaCl and water, pH 7.2; 1.5% agar powder was added to the solid medium.
[0050] D-Allulose was purchased from COFCO Group; Inulin was purchased from Guangdong Quantum High-Tech Biotechnology Co., Ltd.; Fructooligosaccharides were purchased from Guangdong Quantum High-Tech Biotechnology Co., Ltd., under the trade name "Oulido". ® .
[0051] Unless otherwise specified, all other reagents and consumables used in the following examples are commercially available, and the relevant experimental steps are common operations in the field and have technical meanings that can be conventionally understood by those skilled in the art.
[0052] Example 1
[0053] Isolation and identification of Lactobacillus rhamnosus WP502:
[0054] Adult fecal samples were serially diluted with sterile physiological saline under aseptic conditions, spread on MRS agar plates, and incubated at 37°C for 48 hours. Colony morphology was observed visually; single colonies that were round, with neat edges, smooth and raised surfaces, opaque, milky white, bright, and moist and viscous were selected. These colonies were then repeatedly streaked on MRS solid medium for purification. After microscopic observation, preliminary screening and purification were performed. The purified strains were incubated in MRS liquid at 37°C for 16 hours, centrifuged to remove the supernatant, and resuspended in sterile 25% glycerol aqueous solution, then stored at -80°C.
[0055] The selected target strain was cultured in liquid medium, bacterial cells were collected, genomic DNA was extracted, and PCR amplification was performed using universal primers 27F / 1492R to obtain PCR products. The PCR products were then sequenced after gel electrophoresis, and the identified gene sequences were submitted to the NCBI database for BLAST analysis and comparison. Based on the molecular biological identification results, the strain was identified as *Lactobacillus rhamnosus*, named WP502, and deposited.
[0056] The sequence of 16sRNA is shown in SEQ ID No. 1.
[0057] SEQ ID No. 1:
[0058]
[0059] Example 2
[0060] Antagonistic ability of Lactobacillus rhamnosus WP502 against common vaginal pathogens
[0061] Preparation of indicator bacterial suspensions: *Escherichia coli* (Ec), *Staphylococcus aureus* (Sa), and *Salmonella* (St) were activated on LB agar medium, respectively; *Gardnerella vaginalis* (Gv) and *Atobothecus vaginalis* (Av) were activated on BHI agar medium supplemented with 5% defibrinated sheep blood, respectively. Bacterial colonies were picked and added to physiological saline to prepare bacterial suspensions, and the bacterial concentration was adjusted to 10⁻⁶. 8 CFU / mL; Candida albicans was activated and cultured in YPD liquid in shake flasks for 14-16 hours, and the bacterial concentration was adjusted to 10. 8 CFU / mL.
[0062] Antibacterial test: The viable count of the indicator bacteria was diluted to 10⁻¹⁰ using their respective agar-containing culture media. 6 The concentration was on the order of CFU / mL. The culture was then rapidly poured into plates pre-filled with Oxford cups. After the culture medium cooled and solidified, the Oxford cups were removed, and 200 μL of lactic acid bacteria fermentation broth (with a viable count of 10⁻⁶) was injected into each well. 8 After incubation at 37°C overnight (CFU), the diameter of the inhibition zone was measured. The results are shown in Table 1.
[0063] Table 1
[0064]
[0065] The data in Table 1 show that *Lactobacillus rhamnosus* WP502 has a broad-spectrum antibacterial effect against common vaginal pathogens. In particular, it has a very good antibacterial effect against *Gardnerella vaginalis*, the core pathogen of bacterial vaginosis. This indicates that *Lactobacillus rhamnosus* WP502 can exert a broad-spectrum antibacterial effect through multiple means, including fermentation to produce acid, antibiotics and other antibacterial substances, competitive inhibition, immune regulation, inhibition of virulence factors, and maintenance of ecological balance, thus helping to prevent and treat vaginal infections.
[0066] Example 3
[0067] Study on the self-cohesion of Lactobacillus rhamnosus WP502 and its cohesive power with Gardnerella vaginalis
[0068] Experimental strain preparation: After 2 generations of activation, WP502 and GR-1 strains were transferred to 3 generations at a 3% inoculum concentration and cultured for 8 hours. Gardnerella vaginalis (Gv) was activated and cultured on BHI solid medium containing 5% defibrinated sheep blood.
[0069] (1) Determination of cohesion: The cultured WP502 and GR-1 bacterial cultures were centrifuged, the bacterial cells were collected, washed twice with PBS buffer, and then resuspended in PBS to adjust the OD of the strains. 600 The value was 0.6. Each strain was taken into a 40 mL to 50 mL centrifuge tube, mixed well, vortexed for 20 s, and then incubated at 37 °C. After incubation for 2 h, 5 h, 21 h, and 24 h, 2.5 mL of the supernatant was carefully aspirated and the absorbance (At) of the bacterial suspension at 600 nm was measured to calculate the self-cohesion of each strain.
[0070] Cohesive strength (%) = [1 - At / A0] × 100%
[0071] Where A0 is the initial absorbance value, and At is the absorbance value when sampled at 2h, 5h, 21h, and 24h.
[0072] The results are as follows Figure 1 As shown, the self-cohesion of WP502 gradually increased over time, reaching as high as 46.03% at 24 hours, which is significantly stronger than that of commercial strains. This indicates that Lactobacillus rhamnosus WP502 can exert its probiotic effects more stably and for longer in the vagina, such as enhancing the vaginal mucosal barrier function, enhancing immune regulation, better maintaining the vaginal acidic environment, and reducing the risk of vaginitis recurrence.
[0073] (2) Copolymerization assay with Gv: The cultured WP502, GR-1 and Gardnerella vaginalis (Gv) were centrifuged to collect the probiotic and pathogen cells. The cells were washed twice with PBS buffer and then resuspended in PBS. The OD of the probiotics was adjusted to the specified values. 600 The value is 0.6, indicating the OD value of the pathogen. 600 The value was 0.6. After thorough shaking and mixing, the initial OD was measured. 600 The values Ax and Ay were determined. 20 mL of probiotics and 20 mL of pathogens were taken and mixed in a 50 mL centrifuge tube. The mixture was vortexed for 20 s and then incubated at 37 °C. After incubation for 2 h, 5 h, 21 h, and 24 h, 2 mL of the supernatant was carefully aspirated and the absorbance (A) of the bacterial suspension at 600 nm was measured. The cohesive force between the bacterial strain and the pathogen was calculated.
[0074] Cohesive strength (%) = [(Ax+Ay) / 2 - A(x+y)] / [Ax+Ay / 2] × 100%
[0075] Where Ax and Ay represent the initial OD before mixing, respectively. 600 The value, A(x+y), represents the OD of the mixture. 600 value.
[0076] The results are as follows Figure 2As shown, over time, the cohesive force between WP502 and Gv gradually increased, and the cohesive effect was better than that of the control strain GR-1. This indicates that WP502 can effectively prevent pathogens from attaching and multiplying, exert a competitive exclusion effect, inhibit their growth and toxicity, promote their excretion, reduce the concentration of pathogens in the vagina, and reduce the risk of infection.
[0077] Example 4
[0078] Hydrophobicity determination of Lactobacillus rhamnosus WP502 surface
[0079] The activated WP502 and GR-1 bacterial suspensions were centrifuged at 10000×g for 1 min. The bacterial cells were washed twice with PBS solution (pH 7.4), resuspended in PBS, and the bacterial suspension concentration was adjusted to 10. 8 The absorbance of the bacterial suspension was measured at 600 nm (A0=1.0) using CFU / mL. 12 mL of the bacterial suspension was mixed with 4 mL of xylene, vortexed at room temperature (25℃) for 1 min, and then allowed to stand for 10 min. The mixture was then vortexed again for 1 min and allowed to stand at room temperature for 20 min to separate into two layers: a transparent lower aqueous layer and a cell-containing upper solvent layer. The absorbance of the aqueous phase was measured at 600 nm (A1), with the buffer solution serving as a blank control. Surface hydrophobicity was expressed as the percentage of organic solvent adhering to the bacteria.
[0080] Surface hydrophobicity (%) = (1 - A1 / A0) × 100%
[0081] A0 and A1 are the absorbances of the bacterial solution measured at 600 nm before and after mixing with xylene, respectively.
[0082] The results are as follows Figure 3 As shown, the surface hydrophobicity of WP502 is as high as 92.408%, which is significantly stronger than that of the commercial strain GR-1. This indicates that WP502 has a strong potential to adhere to vaginal epithelial cells, thereby protecting the vaginal mucosal barrier from pathogenic microorganisms and exerting its beneficial function.
[0083] Example 5
[0084] Study on the effect of Gv in alleviating bacterial vaginosis in BALB / c mice
[0085] (1) Experimental animals:
[0086] SPF-grade BALB / c female mice (8 weeks old, weighing 18-20g) were purchased from Huazhong Agricultural University in Wuhan and were housed in an environment with 22±2℃, 55%±5% humidity, and 12h light-dark cycle, with free access to food and water.
[0087] (2) Grouping of animals:
[0088] After the mice had adapted to the feeding for one week, they were randomly divided into groups of 6 mice each.
[0089] The study included a healthy control group (NC group), a model group (MC group), and an experimental group. The specific grouping and treatment methods for the experimental group are as follows:
[0090] Experimental Group 1 (referred to as Group S1): The total viable count of the strains in the probiotic preparation was 5 × 10⁻⁶. 9 The strain is a combination of *Lactobacillus rhamnosus* WP502, *Lactobacillus paracasei* LC97, and *Lactobacillus plantarum* Lp18 with a viable count of 1:1:1, and contains 0.5% D-alokulose, 0.5% fructooligosaccharides, and 0.5% inulin.
[0091] Experimental Group 2 (referred to as Group S2): The total viable count of the strains in the probiotic preparation was 5 × 10⁻⁶. 9 The strain is a combination of *Lactobacillus rhamnosus* WP502 and *Lactobacillus paracasei* LC97 with a viable count of 1:1, and contains 0.5% D-allulose, 0.5% fructooligosaccharides, and 0.5% inulin.
[0092] Experimental Group 3 (referred to as Group S3): The total viable count of the strains in the probiotic preparation was 5 × 10⁻⁶. 9 The strain is a combination of *Lactobacillus rhamnosus* WP502 and *Lactobacillus plantarum* Lp18 with a viable count of 1:1, and contains 0.5% D-allulose, 0.5% fructooligosaccharides, and 0.5% inulin.
[0093] Experimental Group 4 (referred to as Group S4): The total viable count of the strains in the probiotic preparation was 5 × 10⁻⁶. 9 The strain is a combination of *Lactobacillus paracasei* LC97 and *Lactobacillus plantarum* Lp18 with a viable count of 1:1, and contains 0.5% D-allulose, 0.5% fructooligosaccharides, and 0.5% inulin.
[0094] Experimental group 5 (designated S5): The total viable count of the strains in the probiotic preparation was 5 × 10⁻⁶. 9 The strain is a combination of *Lactobacillus rhamnosus* WP502, *Lactobacillus paracasei* LC97, and *Lactobacillus plantarum* Lp18 with a viable count of 1:1:1, and contains 0.75% D-alokulose and 0.75% fructooligosaccharides.
[0095] Experimental group 6 (designated S6): The total viable count of the strains in the probiotic preparation was 5 × 10⁻⁶. 9 The strain is a combination of *Lactobacillus rhamnosus* WP502, *Lactobacillus paracasei* LC97, and *Lactobacillus plantarum* Lp18 with a viable count of 1:1:1, and contains 0.75% D-alokulose and 0.75% inulin.
[0096] Experimental group 7 (designated S7): The total viable count of the strains in the probiotic preparation was 5 × 10⁻⁶. 9 The strain is a combination of *Lactobacillus rhamnosus* WP502, *Lactobacillus paracasei* LC97, and *Lactobacillus plantarum* Lp18 with a viable count of 1:1:1, and contains 0.75% fructooligosaccharides and 0.75% inulin.
[0097] Experimental group 8 (designated S8): The viable count of the strain in the probiotic preparation was 5 × 10⁸. 9 CFU / mL, the strain being Lactobacillus rhamnosus WP502.
[0098] The preparation method of the probiotic preparation in the experimental group includes: inoculating the strain into MRS medium, centrifuging the culture medium to obtain bacterial cells, mixing the bacterial cells with a protectant, freeze-drying, and then mixing with prebiotics to obtain the final product.
[0099] (3) Animal modeling:
[0100] Mice in both the model group and the probiotic group were subcutaneously injected with 100 μL of estradiol benzoate to induce estrus. This injection was repeated every 4 days. After estrus, mice in both groups were inoculated vaginally with 20 μL of bacteria at a concentration of 1×10⁻⁶. 10 Gardnerella vaginalis (Gv) at CFU / mL was inoculated once daily for 5 consecutive days until the model was established. The healthy group received an intravaginal injection of 20 μL of physiological saline.
[0101] Model validation criteria: redness and swelling of the vaginal opening accompanied by abundant vaginal discharge. The Gv copy number was calculated by qPCR of the discharge, confirming that the target bacteria had significantly proliferated.
[0102] (4) Animal intervention methods:
[0103] Healthy control and model group mice were injected intravaginally with 20 μL of physiological saline; experimental group mice were inoculated intravaginally with 20 μL of bacteria at a concentration of 5 × 10⁻⁶. 9 A probiotic preparation of CFU / mL was administered once daily for 12 consecutive days.
[0104] (5) Indicator Analysis:
[0105] (5.1) Observation of vaginal opening and vaginal tissue
[0106] Before dissecting the mice, the redness and swelling of the vaginal opening of each group of mice were observed and photographed. After vaginal irrigation, the mice were euthanized by cervical dislocation, and the vaginal tissue was dissected and photographed to observe the redness and swelling of the vaginal tissue.
[0107] The results are as follows Figure 4 As shown, compared with the NC group, the MC group showed slight redness and swelling of the vaginal tissue, accompanied by congestion; indicating that the model was successfully established.
[0108] (5.2) Analysis of Gardnerella vaginalis load in vaginal secretions:
[0109] Mice were vaginally cleansed three times with 300 μL of physiological saline, and the vaginal lavage fluid was collected. The number of Gardnerella vaginalis in the lavage fluid was quantified using qPCR. The upstream and downstream primer sequences used are shown in Table 2. The qPCR reaction system (10 μL) is shown in Table 3. The reaction program was set to 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ annealing for 30 s, for 40 cycles. In another step, the temperature was increased from 65℃ to 95℃ in 0.5℃ increments every 5 s to establish a melting curve. The fluorescence threshold cycle value (CT) was determined for each sample, and the copy number was calculated based on the standard curve (log copies / μL vs. CT value). Each sample was tested in triplicate.
[0110] Table 2
[0111]
[0112] Table 3
[0113]
[0114] The results are as follows Figure 5 , 6 As shown, Gardnerella vaginalis was not detected in the vaginal douche fluid of the NC group, while the Gardnerella vaginalis load in the model group was significantly higher than that in the healthy group, indicating that Gardnerella vaginalis successfully colonized the vagina. After intervention with WP502, the Gardnerella vaginalis load in the probiotic group was significantly reduced, indicating that WP502 can significantly inhibit the growth and reproduction of bacterial vaginosis bacteria in vivo. Moreover, the effect of S1 was better than that of the two-strain combination (S2, S3, S4), and better than that of S5, S6, S7 and the single-strain S8 group.
[0115] (5.3) Pathological analysis of vaginal tissue sections:
[0116] After euthanizing mice by cervical dislocation, vaginal tissues were dissected from mice in the NC, MC, and WP502 groups. Some tissues were fixed by soaking in 10% paraformaldehyde solution (some were directly stored at -80°C for later use), then stained with hematoxylin and eosin (HE), and finally the vaginal tissue structure was observed using an optical microscope.
[0117] like Figure 7 As shown, HE staining of vaginal tissue from mice in the NC group revealed that the vaginal mucosal epithelium was continuous, intact, and smooth, with no large amounts of sloughed vaginal epithelial tissue. The goblet cells in the vaginal mucosa maintained their morphology and structure, were neatly arranged, and had their nuclei located inside the goblet cells, showing no inflammatory cell infiltration. In contrast, the vaginal mucosal epithelium in the MC group was severely damaged, with extensive sloughing of the vaginal mucosal epithelium extending to the lamina propria, accompanied by inflammatory cell infiltration. Compared to the MC group, the vaginal mucosal epithelium in the WP502 group showed less damage, with the vaginal mucosal epithelium gradually becoming continuous, less inflammatory cell infiltration, and a relatively compact lamina propria structure. This indicates that WP502 can effectively promote the recovery of the vaginal mucosa in mice, alleviate inflammatory symptoms in the vaginal tissue, and improve the damaged vaginal mucosal barrier.
[0118] (5.4) Analysis of inflammatory factors (TNF-α, IL-17, IL-6, IL-1β, IL-10, MPO, SIgA):
[0119] Vaginal tissue samples stored at -80℃ were processed strictly according to the instructions of the ELISA kit. The levels of cellular inflammatory factors TNF-α, IL-1β, IL-6, IL-17, IL-10, MPO and SIgA in the vaginal tissue lysate of each group of mice were detected.
[0120] The results are as follows Figure 8 As shown, compared with the NC group, the levels of pro-inflammatory factors TNF-α, IL-1β, IL-6, IL-17 and MPO were significantly increased in the MC group, while the secretion of anti-inflammatory factor IL-10 and immune protein SIgA was significantly reduced. After intervention with various probiotics and prebiotics, the secretion levels of TNF-α, IL-17, IL-6 and MPO in the vagina of mice were significantly reduced, and the secretion level of IL-1β was significantly reduced; at the same time, the secretion levels of anti-inflammatory factor IL-10 and immune protein SIgA were significantly enhanced. This indicates that WP502 can effectively reduce the secretion of pro-inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-17, as well as the MPO content in vaginal tissue. The S1 group is more effective than the S5, S6, S7, S2, S3, S4, and the single-strain S8 group. At the same time, it effectively promotes the secretion of anti-inflammatory factor IL-10 and immune protein SIgA, thereby playing an immunomodulatory role and achieving the goal of improving vaginal health. The S1 group is more effective than the S5, S6, S7, S2, S3, S4, and the single-strain S8 group.
[0121] (5.5) Analysis of vaginal mucosal barrier protein factors
[0122] Vaginal tissue samples stored at -80℃ were processed strictly according to the instructions of the ELISA kit, and the contents of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1 in the vaginal tissues of mice in each group were detected.
[0123] ZO-1, CLDN1, and OCLN proteins are standard markers for assessing barrier integrity. The results of the detection of the levels of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1 in vaginal tissues of mice in each group are shown below. Figure 9 As shown, compared with the NC group, the levels of vaginal mucosal barrier protein factors ZO-1, OCLN, and CLDN1 in the MC group were significantly reduced, indicating severe damage to the vaginal mucosal barrier. However, after intervention with WP502 and various compound probiotic preparations, the levels of vaginal mucosal barrier protein factors were significantly increased, indicating that Lactobacillus rhamnosus WP502 can effectively repair the damaged vaginal mucosal barrier, and the effect of the S1 group was better than that of the S5, S6, S7, S2, S3, S4 and the single strain S8 group.
[0124] (5.6) Vaginal flora analysis
[0125] Total microbial DNA was extracted from the homogenate lysate of mouse vaginal tissue. The V3-V4 region of the 16S rRNA gene was amplified by PCR, followed by paired-end sequencing. The sequencing data underwent quality control and filtering, and cluster analysis was performed to determine phylogenetic relationships. Finally, changes in the mouse vaginal flora were assessed.
[0126] To visualize the overall vaginal microbiota structure, we used PCA to analyze the β-diversity of vaginal flora in each group of mice, such as... Figure 10 As shown, the model group and the control group were completely separated, and the community structure of the Lactobacillus rhamnosus WP502 group was close to that of the control group, indicating that Lactobacillus rhamnosus WP502 can effectively regulate the vaginal microbial community structure of BV mice.
[0127] To more clearly understand the changes in the relative abundance of vaginal microbiota, Figure 11 The study revealed the microbial community structure characteristics at the phylum level in the gut microbiota of each group of mice. Compared with the MC group, WP502 significantly reduced the relative abundance of Proteobacteria and increased the relative abundance of Firmicutes.
[0128] The results of the genus-level taxonomic analysis of the gut microbiota of each group of mice are as follows: Figure 12 and Figure 13As shown, the relative abundance of *Escherichia Shigella* and *Alcaligenes* in the MC group was significantly increased compared to the control group (p<0.001), the relative abundance of the opportunistic pathogen *Enterococcus* was significantly increased (p<0.05), and the relative abundance of the beneficial bacteria *Streptococcus* was significantly decreased (p<0.05). After WP502 intervention, the probiotic group significantly reduced the relative abundance of *Alcaligenes* (p<0.001), significantly reduced the relative abundance of *Escherichia Shigella* and *Enterococcus* (p<0.05), and significantly promoted the increase of the relative abundance of *Streptococcus* (p<0.05). This indicates that WP502 can effectively regulate the vaginal flora and increase the vaginal flora richness in mice with Gv-induced bacterial vaginosis.
[0129] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.
Claims
1. A probiotic agent for repairing damage to the reproductive tract mucosal barrier, characterized in that, The probiotic strains in the probiotic agent include Lacticaseibacillus rhamnosus WP502 strain with accession number CGMCC No.37035, Lacticaseibacillus paracasei LC97 strain with accession number CGMCC No.32421, and Lactiplantibacillus plantarum Lp18 strain with accession number CCTCC NO: M 20231524.
2. The probiotic agent for repairing damage to the reproductive tract mucosal barrier according to claim 1, characterized in that, The ratio of viable counts of the Lactaseibacillus rhamnosus WP502 strain, Lactaseibacillus paracasei LC97 strain, and Lactiplantibacillus plantarum Lp18 strain was (1-4):(1-2):(1-2).
3. The probiotic agent for repairing damage to the reproductive tract mucosal barrier according to claim 1, characterized in that, The probiotic agent also includes prebiotics.
4. The probiotic agent for repairing damage to the reproductive tract mucosal barrier according to claim 3, characterized in that, The prebiotic content in the probiotic agent is 1-10% by mass.
5. The probiotic agent for repairing damage to the reproductive tract mucosal barrier according to claim 3, characterized in that, The prebiotics include D-alloxanose, fructooligosaccharides, and inulin.
6. The probiotic agent for repairing damage to the reproductive tract mucosal barrier according to claim 5, characterized in that, The mass ratio of D-allulose, fructooligosaccharide and inulin is (1-3):(1-3):(1-3).
7. A method for preparing a probiotic agent for repairing damage to the reproductive tract mucosal barrier according to any one of claims 1-6, characterized in that, The preparation method includes: inoculating the bacterial strains into a culture medium, culturing, collecting the bacterial sludge by centrifugation, mixing the bacterial sludge with a protectant, and freeze-drying to obtain the final product.
8. The use of the probiotic agent for repairing damage to the reproductive tract mucosal barrier according to any one of claims 1-6 in the preparation of a medicine having the effect of repairing damage to the reproductive tract mucosal barrier.
9. The use of the probiotic agent for repairing damage to the reproductive tract mucosal barrier according to any one of claims 1-6 in the preparation of a medicine for treating bacterial vaginosis.