Compositions for alleviating vaginitis by simultaneously activating immunomodulatory and antioxidant pathways

Abstract

The application discloses a composition for relieving vaginitis by simultaneously activating immune regulation and antioxidant pathways, and belongs to the technical field of microorganisms. The application screens a composite strain containing a strain of Paracaseolovis casei CCFM1316 and a strain of Paracaseolovis casei CCFM1317, and the composition containing the composite strain has the effect of repairing immune disorders, oxidative damage and other related symptoms caused by vaginitis, which is specifically embodied in: regulating the immune response of the mouse vagina; activating the TLR-4 / MyD88 pathway, and reducing the IL-1beta concentration of the vaginal tissue to 29.79 ng / L; improving the oxidative stress of the mouse vagina; and increasing the Nrf2 of the vaginal tissue to 36.58 ng / L. Therefore, the composition of the Paracaseolovis casei CCFM1316 and the Paracaseolovis casei CCFM1317 has great application prospect in products for improving and repairing the inflammatory response and oxidative damage caused by vaginitis.

Description

Technical Field

[0001] This invention relates to a Lactobacillus paracasei composition that can alleviate immune disorders and oxidative damage caused by vaginitis, belonging to the field of microbial technology. Background Technology

[0002] Vaginitis is a common vaginal infection in women of childbearing age. Pathogens such as bacteria and fungi invade the vaginal cavity and colonize it, causing damage to the vaginal environment during their reproduction. Clinical cases are characterized by a high recurrence rate and atypical clinical symptoms, making it easy to miss in clinical practice. Treatment usually involves continued use of antibacterial agents.

[0003] Vaginal epithelial cells and immune cells circulating in the vaginal tissue are the first to come into contact with the pathogenic microorganism. The pathogen molecules bind to Toll-like receptors (TLRs), activating the myeloid differentiation factor 88 (MyD88) signaling pathway, secreting pro-inflammatory cytokines such as IL-1β, triggering an immune response of the vaginal mucosa to Gardnerella vaginalis, accompanied by tissue inflammation. Simultaneously, Gardnerella vaginalis, invading the vaginal environment, adheres to reproductive tract epithelial cells, causing mitochondrial damage and producing large amounts of reactive oxygen species (ROS), leading to an imbalance in the body's redox system and causing oxidative damage.

[0004] Currently, treatment primarily involves the use of anti-anaerobic drugs, mainly including nitroimidazoles (metronidazole, tinidazole) and lincomycins (clindamycin). However, antibiotics cannot regulate the vaginal immune response or repair damage caused by oxidative stress, increasing the risk of recurrent vaginitis.

[0005] Studies have shown that adding live lactobacillus capsules to the treatment of recurrent bacterial vaginosis can significantly reduce the recurrence rate. Live lactobacillus preparations can effectively improve the local microecological environment, achieving the goal of treating bacterial vaginosis with significant efficacy. Furthermore, a comparison of the efficacy of three single-strain probiotics and compound probiotic formulations containing the corresponding single strains revealed that both single and compound probiotics effectively promoted the immune function of mice, with the compound probiotics showing a more significant therapeutic effect. Therefore, the simultaneous use of multiple strains may produce a synergistic effect, enhancing the survival ability of the strains and enabling them to exert a sustained, stable, and comprehensive regulatory effect on the body.

[0006] Currently, most probiotic compositions for treating vaginitis are based on Lactobacillus curvatureii, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus reuteri, or Lactobacillus rhamnosus (CN111647525A, CN110016442A, CN114480191A). At the same time, there is a lack of probiotic agents that can alleviate oxidative damage caused by vaginitis, as well as those that can alleviate vaginitis by activating immune regulatory pathways and antioxidant capacity. Summary of the Invention

[0007] To address the shortcomings of the prior art, this invention provides a composition that simultaneously activates immune regulation and antioxidant pathways to alleviate vaginitis. The aim is to solve the technical problem of the lack of probiotics in the prior art that alleviate vaginitis by improving oxidative damage caused by vaginitis.

[0008] The first technical solution provided by this invention is a strain of Lacticaseibacillus paracasei CCFM1316, the accession number of which is GDMCC No:63712.

[0009] The second technical solution provided by the present invention is a composition containing Lactobacillus paracasei CCFM1316 and Lactobacillus paracasei CCFM1317 as described in the first technical solution; the accession number of Lactobacillus paracasei CCFM1317 is GDMCC No:63713.

[0010] In some embodiments, the composition is a compound bacterial suspension containing Lactobacillus paracasei CCFM1316 and Lactobacillus paracasei CCFM1317.

[0011] In some embodiments, the content of the composite bacterial solution in the composition is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0012] Furthermore, the content of the compound bacterial solution in the composition is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0013] In some embodiments, the ratio of *Lactobacillus paracasei* CCFM1316 to *Lactobacillus paracasei* CCFM1317 in the compound bacterial solution is 1:1.

[0014] The third technical solution provided by the present invention is a product containing Lactobacillus paracasei CCFM1316 as described in the first technical solution or the composition described in the second technical solution.

[0015] In some embodiments, the content of *Lactobacillus paracasei* CCFM1316 in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0016] Furthermore, the content of *Lactobacillus paracasei* CCFM1316 in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0017] In some embodiments, the product is food, medicine, or hygiene product.

[0018] Furthermore, the medicine comprises the above-described composition and a pharmaceutically permissible carrier.

[0019] Furthermore, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, humectants, disintegrants, lubricants, and flavoring agents.

[0020] Furthermore, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0021] Furthermore, the medicines include enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0022] Furthermore, the hygiene products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and antibacterial / bacteriostatic washes for women.

[0023] In some embodiments, the content of the compound bacterial solution in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0024] Furthermore, the content of the compound bacterial solution in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0025] The four technical solutions provided by this invention are the application of Lactobacillus paracasei CCFM1316 described in the first technical solution and the composition described in the second technical solution in the preparation of products for relieving and / or treating vaginitis.

[0026] In some embodiments, the effects of alleviating and / or treating vaginitis include activating the TLR-4 / MyD88 pathway, reducing IL-1β levels in vaginal tissue, and increasing Nrf2 levels in vaginal tissue, thereby improving oxidative stress in the mouse vagina. Nuclear factor-erythrocyte-associated factor 2 (Nrf2) is an important antioxidant transcription factor. Under normal conditions, Nrf2 remains at low levels in the cytoplasm. Probiotics, by activating the Nrf2 signaling pathway, can increase the expression levels of the Nrf2 gene and protein, thereby increasing the anti-inflammatory and antioxidant capacity of cells.

[0027] In some embodiments, the product is food, medicine, or hygiene product.

[0028] Furthermore, the medicine comprises the above-described composition and a pharmaceutically permissible carrier.

[0029] Furthermore, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, humectants, disintegrants, lubricants, and flavoring agents.

[0030] Furthermore, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0031] Furthermore, the medicines include enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0032] Furthermore, the hygiene products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton pads, vaginal washes, and antibacterial / bacteriostatic washes for women.

[0033] In some embodiments, the content of the compound bacterial solution in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0034] Furthermore, the content of each of the compound strains in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0035] Compared with the prior art, the present invention has the following technical effects; the present invention screened and obtained a compound bacterial solution containing one strain of Lactobacillus paracasei CCFM1316 and one strain of Lactobacillus paracasei CCFM1317, and the composition containing the compound strain has the effect of repairing the immune disorders, oxidative damage and other related symptoms caused by vaginitis, specifically manifested in: (1) reducing the shedding of vaginal epithelial cells in mice with vaginitis; (2) regulating the vaginal immune response in mice: activating the TLR-4 / MyD88 pathway, and reducing the concentration of IL-1β in vaginal tissue to 29.79 ng / L; (3) improving vaginal oxidative stress in mice: increasing Nrf2 in vaginal tissue to 36.58 ng / L; (4) improving the pathological features of the vagina in mice and reducing the pathogenic bacterial load. Therefore, the combination of Lactobacillus paracasei CCFM1316 and Lactobacillus paracasei CCFM1317 has great application potential in products that improve and repair inflammatory responses and oxidative damage caused by vaginitis.

[0036] Preservation of biological materials

[0037] The Lacticaseibacillus paracasei CCFM1316 provided by this invention was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No:63712, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0038] The Lacticaseibacillus paracasei CCFM1317 provided by this invention was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:63713, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0039] Figure 1 Animal experiment design flowchart.

[0040] Figure 2 Effect of Lactobacillus paracasei on the concentration of the vaginal pro-inflammatory factor IL-1β; **p<0.01, ***p<0.001, ****p<0.0001.

[0041] Figure 3: Effect of Lactobacillus paracasei on vaginal inflammatory pathways; (a) Effect on TLR-4 mRNA expression; (b) Effect on MyD88 mRNA expression; **p<0.01, ***p<0.001, ****p<0.0001.

[0042] Figure 4 Effect of Lactobacillus paracasei on vaginal Nrf2 concentration; **p<0.01, ***p<0.001, ****p<0.0001.

[0043] Figure 5 Image showing vaginal epithelial cell shedding; *p<0.05, ****p<0.0001.

[0044] Figure 6 : Colonization of Gardnerella vaginalis in the vagina; Gardnerella vaginalis load in the vagina of BV mice on day 7 and day 17; a and b represent significant differences between different groups on day 17; p<0.05.

[0045] Figure 7 : Histopathological evaluation of mouse vaginal tissue. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] The culture media involved in the following examples are as follows:

[0048] MRS medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL, pH 6.2–6.4.

[0049] BHI medium: tryptone 10.0 g / L, bovine heart extract 17.5 g / L, sodium chloride 5.0 g / L, glucose 3.0 g / L, disodium hydrogen phosphate dodecahydrate 2.5 g / L, yeast extract 10.0 g / L, maltose 1.0 g / L, pH 7.2–7.4; after cooling to about 55°C, add 10% sterile fetal bovine serum.

[0050] The experimental animals and bacterial strains involved in the following examples are as follows:

[0051] SPF - level BALB / c mice, female, 7 weeks old, weighing 18 - 20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License Number: SCXK(Beijing)2012 - 0001). During the feeding period, each mouse had free access to water and was fed with SPF mouse feed (Co 60 disinfection). Feeding environment: semi - circular lighting for half of the day and night, temperature 22 - 24 °C, humidity about 45% - 55%.

[0052] Gardnerella vaginalis was Gardnerella vaginalis ATCC 14018, purchased from the GDMCC (Guangdong Microbial Culture Collection Center) of the Guangdong Institute of Microbiology.

[0053] Lactobacillus gasseri QJSWX195M1 was preserved in the Food Microbial Culture Collection Center of Jiangnan University.

[0054] The bacterial suspensions involved in the following examples are as follows:

[0055] Lactobacillus paracasei CCFM1316 and CCFM1317 bacterial suspensions: Lactobacillus paracasei CCFM1316 and CCFM1317 were inoculated into MRS liquid medium at an inoculation amount of 2%, and cultured in an incubator at 37 °C for 24 h, and then the concentration of the bacterial suspension was adjusted to ~10 9 CFU / mL.

[0056] Gardnerella vaginalis suspension: Gardnerella vaginalis strain ATCC 14018 was cultured in BHI medium in an incubator at 37 °C for 24 h, and the concentration of the bacterial suspension was adjusted to 10 10 CFU / mL.

[0057] The animal models and groupings involved in the following examples are as follows:

[0058] Table 1 Animal model experimental plan and grouping

[0059] Grouping Inducing estrus Induction time of Gardnerella vaginalis infection Probiotic intervention time Blank control - - - vaginitis model Days 1-3 Days 3-7 Days 8-17 Lactobacillus paracasei CCFM1316 Days 1-3 Days 3-7 Days 8-17 Lactobacillus paracasei CCFM1317 Days 1-3 Days 3-7 Days 8-17 Composition Days 1-3 Days 3-7 Days 8-17

[0060] The mice were randomly divided into 5 groups according to their body weights. Referring to Table 2, all groups of mice were normally fed throughout the experiment.

[0061] Construction of the vaginitis animal model: SPF - level BALB / c mice, female, 7 weeks old, weighing 18 - 20 g were selected. After the adaptation period, the mice were injected with estradiol valerate for three consecutive days for induced estrus treatment. They were continuously infected with Gardnerella vaginalis for 5 days, once a day. The specific operation method of the infection was to prepare a 10 10 CFU / mL Gardnerella vaginalis suspension, with a dose of 20 μL / mouse. Use a pipette tip to suck 20 μL of the bacterial suspension and slowly inject it into the vagina of the mouse. Then hold the mouse upside down for 1 - 2 minutes and put it back into the cage.

[0062] Mice with a vaginitis model were divided into a model group and an intervention group. The intervention group received 100 μL of a 10% concentration of [unspecified substance] via gavage. 9 The probiotic suspension contained CFU / mL, wherein the composition was a 1:1 mixture of Lactobacillus paracasei CCFM1316 and Lactobacillus paracasei CCFM1317 bacterial suspensions. The model group underwent only Gardnerella vaginalis infection without any subsequent intervention, while the intervention group underwent Gardnerella vaginalis infection and received probiotics as a subsequent intervention. In addition, a blank control group was set up, in which the blank control group did not undergo Gardnerella vaginalis infection and did not receive probiotics as a subsequent intervention.

[0063] Both the vaginitis model group and the intervention group underwent subcutaneous injection of 100 μL of estradiol valerate solution (0.5 mg estradiol valerate dissolved in 100 μL of filtered and sterilized sesame oil) into the neck for 3 consecutive days (days 1-3) to induce estrus. They were then subjected to Gardnerella vaginalis infection for 5 consecutive days (days 3-7). The probiotic intervention group was further treated with *Lactobacillus paracasei* CCFM1316, *Lactobacillus paracasei* CCFM1317, and a combination thereof for 10 consecutive days (days 8-17). The specific procedure for all interventions was as follows: each group of mice was administered 100 μL of a 10% concentration solution via gavage once daily. 9 CFU / mL Lactobacillus resuspension (days 8-17). At the end of the intervention (day 17), 50 μL of phosphate buffer solution was aspirated from the mouse vagina using a pipette tip, resulting in a final collection of 300 μL of vaginal irrigation fluid for subsequent analysis of vaginal epithelial cell shedding. Simultaneously, on day 18, all experimental mice were euthanized, and vaginal and skin tissues were dissected for subsequent histopathological analysis, detecting the expression of inflammatory factors (IL-1β), immune pathway (TLR-4 / MyD88), antioxidant factor (Nrf2) secretion, and antioxidant pathway (Keap1 / Nrf2) expression in the vaginal tissue. The probiotic combination effect was calculated using the Jin Zhengjun Q-value method: Q = E a+b / (E a +E b -E a ×E b ), where E a+b E represents the rate of change of the measured index value when A and B are combined. a and E b These are the rate of change of the detected index values ​​when A and B act alone, respectively. In the formula, the numerator represents the "measured combined effect", the denominator represents the "expected combined effect", and Q is the ratio of the two. Q < 0.85 indicates an antagonistic effect, 0.85 ≤ Q < 1.15 indicates an additive effect, and Q ≥ 1.15 indicates a synergistic effect (Zhao Wenge et al., "Evaluation of the Synergistic Effect of Matrine Combined with Doxorubicin on Human Breast Cancer Cells by Jin Zhengjun Q-value Method", published in 2018).

[0064] Example 1: Isolation and Identification of Lactobacillus paracasei CCFM1316 and Lactobacillus paracasei CCFM1317

[0065] The specific steps are as follows:

[0066] 1. Screening

[0067] The samples were obtained from vaginal secretions of healthy adult women. After pretreatment, the samples were stored in 30% glycerol at -80°C. After thawing, the samples were mixed and 0.5 mL of the sample was added to 4.5 mL of physiological saline. The samples were then serially diluted with physiological saline containing 9 g / L. The appropriate serial dilutions were plated on MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus paracasei* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium for enrichment and preserved in 30% glycerol to obtain *Lactobacillus paracasei* CCFM1316 and CCFM1317. The typical colonies of *Lactobacillus paracasei* were round, milky white, smooth, and raised.

[0068] 2. Identification

[0069] The genomes of strains CCFM1316 and CCFM1317 were extracted, and the 16S rDNA of the strains was amplified and sequenced (the nucleotide sequence of the 16S rDNA amplified from strain CCFM1316, extracted by Shanghai Meiji Biomedical Technology Co., Ltd., is shown in SEQ ID NO.1, and that of CCFM1317 is shown in SEQ ID NO.2). The nucleotide sequence of the amplified 16S rDNA of the strains was then compared with the nucleic acid sequence in NCBI. The results showed that the strains were Lacticaseibacillus paracasei, and named Lacticaseibacillus paracasei CCFM1316 and Lacticaseibacillus paracasei CCFM1317.

[0070] Example 2: Expression of the inflammatory factor IL-1β in mouse vaginal tissue

[0071] 20 mg of vaginal tissue from five groups of mice was collected for cytokine determination. The vaginal tissue was homogenized in 180 μL of pre-chilled PBS. The samples were centrifuged at 3000 rpm for 15 min at 4°C. The supernatant from the vaginal tissue was then used to determine the IL-1β concentration according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.).

[0072] The results are as follows Figure 2The study showed the concentration of the inflammatory factor IL-1β in vaginal tissue. The results indicated that, compared to the control group (22.92 ng / L), the IL-1β secreted by the vaginal tissue of the model group mice (41.82 ng / L) was significantly increased (p<0.0001). After probiotic intervention, the IL-1β levels of CCFM1316 (p<0.01) and the composition (p<0.001) were significantly decreased. The IL-1β concentrations of *Lactobacillus paracasei* CCFM1316, *Lactobacillus paracasei* CCFM1317, and the composition after intervention were 34.64, 41.40, and 29.79 ng / L, respectively, with a Q value of 1.60, demonstrating a synergistic effect. Therefore, the composition can synergistically reduce the level of inflammatory factors in vaginal tissue and promote the vaginal tissue's resistance to damage from inflammatory factors.

[0073] Example 3: Expression of TLR-4 / MyD88 mRNA in mouse vaginal tissue, an immune pathway

[0074] Total RNA was extracted from vaginal tissues of five groups of mice using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were calculated, and 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit. The reaction mixture was prepared using ChamQ Universal SYBR qPCRMaster Mix for quantitative real-time PCR. GAPDH was used as an internal control gene, based on 2... -ΔΔCt The expression level of the target gene was calculated using the method described in Table 2.

[0075] Table 2 Primers and Sequences

[0076]

[0077] The results are as follows Figure 3 (a) The expression level of TLR-4 mRNA in the control group was 1, and the expression level in the model group was 1.91, which was significantly higher than that in the control group (p<0.001). The expression levels of TLR-4 mRNA in Lactobacillus paracasei CCFM1316, Lactobacillus paracasei CCFM1317, and after the combination intervention were 0.53, 1.10, and 0.78, respectively, all of which significantly downregulated the expression of TLR-4. Figure 3(b) The results showed that, with the expression level of MyD88 mRNA in the control group as 1, the expression level in the model group was 3.13, significantly higher than that in the control group (p<0.001). The expression levels of MyD88 mRNA after intervention with *Lactobacillus paracasei* CCFM1316, *Lactobacillus paracasei* CCFM1317, and the combination were 0.94, 2.50, and 0.76, respectively. Both *Lactobacillus paracasei* CCFM1316 (p<0.001) and the combination (p<0.0001) significantly downregulated the expression of MyD88 mRNA, and the Q value of the combination was 1.05, indicating an additive effect. In summary, probiotic intervention can downregulate the expression of immune pathway-related targets in vaginal tissue, and the combination can achieve an additive effect. Furthermore, it simultaneously downregulates the expression of two targets in the inflammatory pathway, demonstrating the potential to regulate immune pathways and alleviate vaginal inflammatory damage.

[0078] Example 4: Secretion of the antioxidant factor Nrf2 in mouse vaginal tissue

[0079] Five groups of mice were treated with 20 mg of vaginal tissue for cytokine determination. The vaginal tissue was homogenized in 180 μL of pre-chilled PBS. The samples were centrifuged at 3000 rpm for 15 min at 4°C. The supernatant from the vaginal tissue was then used to determine the Nrf2 concentration according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.).

[0080] The results are as follows Figure 4 The concentration of the antioxidant Nrf2 in vaginal tissue was shown. The results indicated that, compared to the control group (20.48 ng / L), the Nrf2 secreted by the vaginal tissue of the model group mice (15.95 ng / L) was significantly decreased (p<0.001). The Nrf2 concentrations of *Lactobacillus paracasei* CCFM1316, *Lactobacillus paracasei* CCFM1317, and the combination intervention were 30.29, 40.09, and 36.58 ng / L, respectively. The Nrf2 content significantly increased after probiotic intervention (p<0.0001), enhancing the antioxidant capacity of vaginal tissue.

[0081] Example 5: Shedding of vaginal epithelial cells in the mouse vagina

[0082] Ten μL of sample solution was taken from the vaginal irrigation fluid of each of the five groups of mice on day 17, transferred to a glass slide, and gently smeared using the outer wall of a pipette tip. The smears were preserved with a fixative and stained using the Diff-Quik staining method (a rapid staining method modified from Wright's staining). Under a microscope, five fields of view were captured from each sample (one mouse), and epithelial cells were counted from each image to determine the mean. Epithelial cell shedding was evaluated under a 400x optical microscope.

[0083] like Figure 5 As shown, the model group exhibited severe epithelial cell shedding (39.33±2.67 cells / field), while the blank control group had approximately 10.67 cells per field. Compared to the model group, *Lactobacillus paracasei* CCFM1316, *Lactobacillus paracasei* CCFM1317, and the combined formulation significantly alleviated vaginal epithelial cell shedding (p<0.0001), with approximately 23.25, 26.00, and 22.67 cells per field in each group, respectively. The combined formulation significantly improved vaginal epithelial cell shedding compared to *Lactobacillus paracasei* CCFM1317 (p<0.05), repairing damage caused by vaginitis.

[0084] Example 6: Gardnerella vaginalis colonization in mouse vagina

[0085] Using a pipette tip, a certain amount of phosphate buffer solution was aspirated and expelled from the vaginas of five groups of mice to obtain 300 μL of vaginal lavage fluid samples. Gardnerella vaginalis and Lactobacillus were counted using qPCR. The entire experimental period was 17 days, with two samplings. The first sampling was on day 7 (the first day after infection ended) as a colonization test for Gardnerella vaginalis, and the second sampling was on day 17 after the intervention to quantify the load of Gardnerella vaginalis and Lactobacillus. First, DNA was extracted from the vaginal lavage fluid using the Soil Rapid DNA Rotation Kit (MP Biomedical, USA) and the QIA Quick Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions. Subsequently, Gardnerella vaginalis and Lactobacillus gasseri were quantitatively detected using qPCR. Primers were selected based on the bacterial 16S rRNA sequence. The reaction mixture (10 μL) included 5 μL of 2×ChamQUniversal SYBR qPCR Master. Mixture (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL template DNA (10 ng / μL), 0.5 μL forward and reverse primers (10 μM each), and 3 μL double-distilled water. Thermal cycling conditions were: initial denaturation at 95°C for 30 s; followed by 95°C for 5 s and 60°C for 30 s, repeated 40 times. In another step, 95°C for 10 s, increasing from 65°C to 95°C in 0.5°C increments every 5 s, was used to establish a melting curve. The threshold cycle value (CT) was determined, and the copy number was calculated based on the standard curve (Log copies / μL vs. CT value). Each sample was tested in triplicate.

[0086] Table 3. Species-specific primers for real-time quantitative PCR detection of Gardnerella vaginalis.

[0087]

[0088] from Figure 6The results showed that, except for the blank control group, approximately 12.511 g copies / μL of Gardnerella vaginalis could be detected in mice on day 7, indicating successful colonization of Gardnerella vaginalis. In the blank control group, Gardnerella vaginalis was not detected throughout the experiment. Several days after probiotic intervention, compared with the model group, the colonization of Gardnerella vaginalis in both the CCFM1316 and CCFM1317 groups was reduced. The combined formula significantly reduced Gardnerella vaginalis colonization (p<0.01).

[0089] Example 7: Histopathological Analysis of Mouse Vaginal Tissue

[0090] Five groups of mice were sacrificed and their vaginas were removed. A portion of the vaginal tissue was used for histopathological examination. The vaginal tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5 mm thick sections, and stained with hematoxylin and eosin (H&E). The vaginal tissue samples were observed at 40x magnification using a pathological slide scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary).

[0091] HE staining of mouse vaginal tissue can effectively assess the inflammation status of vaginal tissue in different groups of mice. Figure 7 As shown, the vaginal epithelium in the blank control group was smooth and continuous, with an intact tissue structure and no obvious inflammatory cell infiltration. In the model group, the vaginal mucosal epithelium showed poor continuity, with superficial cell erosion forming pores, submucosal stroma congestion, and a large number of inflammatory cells infiltrating the epithelium and stroma. Connective tissue hyperplasia was observed in the dermis, with numerous fibroblasts and collagen fibers interwoven. After a period of appropriate probiotic intervention, epithelial continuity was restored, inflammatory cell infiltration was reduced, and submucosal stroma congestion was improved. The connective tissue hyperplasia of CCFM1316 and its combination was significantly improved, demonstrating a good ability to restore damaged vaginal tissue.

[0092] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the examples described above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A composition, characterized in that, Contains Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1316 and Lactobacillus paracasei ( Lacticaseibacillus paracasei The Lactobacillus paracasei CCFM1317 has the accession number GDMCC No:63713, and the Lactobacillus paracasei CCFM1316 is deposited at the Guangdong Provincial Center for Microbial Culture Collection with the accession number GDMCC No:63712.

2. The composition according to claim 1, characterized in that, The composition is a compound bacterial solution containing Lactobacillus paracasei CCFM1316 and Lactobacillus paracasei CCFM1317.

3. The composition according to claim 2, characterized in that, The content of the complex bacteria liquid in the composition is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

4. The composition according to claim 2, characterized in that, In the compound bacterial solution, the ratio of Lactobacillus paracasei CCFM1316 to Lactobacillus paracasei CCFM1317 is 1:

1.

5. A product comprising the composition according to any one of claims 1 to 4, characterized in that, The products mentioned are food and medicine.

6. Use of the composition according to any one of claims 1 to 4 in the preparation of a medicament for relieving and / or treating vaginitis.

7. The application according to claim 6, characterized in that, The effects of alleviating and / or treating vaginitis include activating the TLR-4 / MyD88 pathway, reducing IL-1β levels in vaginal tissue, and increasing Nrf2 levels in vaginal tissue, thereby improving vaginal oxidative stress in mice.

8. The use of the composition according to any one of claims 1 to 4 in the preparation of food.

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