Lactobacillus rhamnosus for improving oxidative damage and alleviating vaginitis

Abstract

The application discloses a lactobacillus rhamnosus capable of improving oxidative damage to relieve vaginitis, and belongs to the technical field of microorganisms. The lactobacillus rhamnosus has the effect of repairing oxidative damage related symptoms caused by vaginitis, and further improves intestinal health problems caused by vaginitis, specifically embodied in: reducing cell Keap1 mRNA expression, increasing cell supernatant Nrf2 content; improving mouse vaginal oxidative stress: activating the Keap1 / Nrf2 pathway, increasing the Nrf2 level of vaginal tissue; enhancing the antioxidant capacity of the mouse intestinal tract: reducing the Keap1 mRNA level of colon tissue; improving the pathological characteristics of the mouse vagina and reducing the pathogenic bacterial load. Therefore, the lactobacillus rhamnosus CCFM1315 has great application prospect in products for improving and repairing the vaginal oxidative damage problems caused by vaginitis.

Description

Technical Field

[0001] This invention relates to a type of Lactobacillus rhamnosus that can improve oxidative damage and alleviate vaginitis, belonging to the field of microbial technology. Background Technology

[0002] Vaginal infections, caused by the invasion and abnormal proliferation of facultative anaerobic bacteria such as Gardnerella vaginalis and anaerobic bacteria, are a common vaginal infection in women of reproductive age. They are characterized by a high recurrence rate and atypical clinical symptoms, making them easily missed in clinical practice.

[0003] Gardnerella vaginalis, which invades the vaginal environment, adheres to the epithelial cells of the reproductive tract and secretes toxins, causing damage to mitochondria. The mitochondrial respiratory chain (ETC) is the main site of reactive oxygen species (ROS) production. Electrons are leaked during ETC transport, and these leaked electrons are directly transferred to oxygen, producing a large amount of ROS. This causes an imbalance in the body's redox reaction system, resulting in oxidative damage.

[0004] Currently, the main treatment for vaginitis involves the use of anti-anaerobic drugs. However, antibiotics cannot repair the vaginal environment damaged by oxidative stress, resulting in a persistently high risk of vaginitis recurrence. Studies have shown that probiotic capsules can effectively improve the local microecological environment and significantly reduce the recurrence rate in the treatment of recurrent bacterial vaginosis.

[0005] In 1989, the Institute of Microecology at Dalian Medical University isolated and screened a strain of Lactobacillus DM8909 from vaginal secretions of healthy women. Through a series of experiments ("Study on the Inhibition of Vaginal Infection in Mice by Lactobacillus DM8909", Chinese Journal of Microecology, 2003; "Immunomodulatory Effect of Peptidoglycan in the Cell Wall of Lactobacillus DM8909 on Vaginal Infection in Mice", Chinese Journal of Microecology, 2013) and clinical data ("Phase II Clinical Trial Study on the Treatment of Bacterial Vaginosis by Lactobacillus delbrueckii DM8909", Chinese Journal of Microecology, 2001), it was shown that Lactobacillus delbrueckii DM8909 has good probiotic properties, regulates the balance of vaginal flora, and effectively treats bacterial vaginosis.

[0006] Furthermore, some patent applications have disclosed the inhibitory ability of *Lactobacillus rhamnosus* against vaginitis pathogens (CN110656060A, CN114480191A, CN110016442B), its ability to regulate vaginal flora (CN112402463B, CN112760247B), and its ability to regulate vaginal immunity (CN110339216A). However, the repair of oxidative damage caused by vaginitis by these strains is unknown, and their efficacy in treating intestinal problems caused by vaginitis is also unclear. Therefore, there is currently a lack of *Lactobacillus rhamnosus* strains that can be clearly used to alleviate vaginitis and intestinal health problems in mice with vaginitis. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides a strain of Lactobacillus rhamnosus that can improve oxidative damage and help alleviate vaginal inflammation through oral administration. The purpose is to solve the technical problem of the lack of Lactobacillus rhamnosus that can be clearly used to alleviate vaginitis and intestinal health problems in mice with vaginitis.

[0008] The first technical solution provided by this invention is a strain of Lactobacillus rhamnosus CCFM1315, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with the accession number GDMCC No:63711.

[0009] The second technical solution provided by the present invention is a microbial preparation containing *Lactobacillus rhamnosus* CCFM1315 as described in the first technical solution.

[0010] In some embodiments, the content of *Lactobacillus rhamnosus* CCFM1315 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0011] Furthermore, the content of *Lactobacillus rhamnosus* CCFM1315 in the microbial preparation is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0012] The third technical solution provided by the present invention is a product containing the *Lactobacillus rhamnosus* CCFM1315 described in the first technical solution or the microbial preparation described in the second technical solution.

[0013] In some embodiments, the product is food or medicine.

[0014] Furthermore, the medicine comprises the above-described composition and a pharmaceutically permissible carrier.

[0015] Furthermore, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, humectants, disintegrants, lubricants, and flavoring agents.

[0016] Furthermore, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0017] Furthermore, the medicines include enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0018] In some embodiments, the content of *Lactobacillus rhamnosus* CCFM1315 in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0019] Furthermore, the content of *Lactobacillus rhamnosus* CCFM1315 in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0020] The four technical solutions provided by this invention are the application of Lactobacillus rhamnosus CCFM1315 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of products for relieving and / or treating vaginitis.

[0021] In some embodiments, the effects of alleviating and / or treating vaginitis include improving vaginal oxidative stress in mice by activating the Keap1 / Nrf2 pathway in vaginal tissue and increasing Nrf2 levels in vaginal tissue.

[0022] In some embodiments, the effects of relieving and / or treating vaginitis include improving the antioxidant capacity of colonic tissue by downregulating Keap1 mRNA expression in colonic tissue.

[0023] Nuclear factor-erythrocyte-associated factor 2 (Nrf2) is an important antioxidant transcription factor. Under normal circumstances, Nrf2 is kept at a low level in the cytoplasm by binding to KELCH-like ECH-associated protein 1 (Keap1). Probiotics can increase the expression of Nrf2 gene and protein by activating the Nrf2 signaling pathway, thereby increasing the anti-inflammatory and antioxidant capacity of cells.

[0024] In some embodiments, the product is food or medicine.

[0025] Furthermore, the drug comprises the aforementioned strains or preparations, as well as pharmaceutically permissible carriers.

[0026] Furthermore, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, humectants, disintegrants, lubricants, and flavoring agents.

[0027] Furthermore, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0028] Furthermore, the medicines include enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0029] Compared with existing technologies, the present invention has the following technical effects: The present invention provides a strain of Lacticaseibacillus rhamnosus CCFM1315, which has the effect of repairing symptoms related to oxidative damage caused by vaginitis and further improving intestinal health problems caused by vaginitis. Specifically, it is reflected in: (1) reducing Keap1 mRNA expression in cells and increasing Nrf2 content in cell supernatant; (2) improving vaginal oxidative stress in mice: activating the Keap1 / Nrf2 pathway and increasing Nrf2 levels in vaginal tissue; (3) enhancing the antioxidant capacity of mouse intestines: downregulating Keap1 mRNA levels in colon tissue; (4) improving vaginal pathological features in mice and reducing pathogenic bacterial load. Therefore, Lacticaseibacillus rhamnosus CCFM1315 has great application prospects in products that improve and repair vaginal oxidative damage caused by vaginitis.

[0030] Preservation of biological materials

[0031] The Lacticaseibacillus rhamnosus CCFM1315 provided by this invention was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:63711, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0032] Figure 1 Effects of different bacterial lysates on Nrf2 content and Nrf2 / SOD1 mRNA expression in the culture supernatant of vaginal epithelial cells VK2 / E6E7; compared with the model group, **p<0.01, ***p<0.001.

[0033] Figure 2 Animal experiment design flowchart.

[0034] Figure 3 Effect of *Lactobacillus rhamnosus* on vaginal oxidative response; (a) Nrf2 concentration; (b) Keap1 mRNA expression. Compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0035] Figure 4 Effect of Lactobacillus rhamnosus on the concentration of Nrf2, a product of colonic oxidation reaction; ****p<0.0001.

[0036] Figure 5:Colonization map of Gardnerella vaginalis in the vagina; Gardnerella vaginalis load in the vagina of BV mice on the 7th and 17th days; a and b represent significant differences between different groups on the 17th day (p<0.01).

[0037] Figure 6 :Histopathological evaluation map of mouse vaginal tissue. Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to specific embodiments and the attached drawings.

[0039] The culture media involved in the following embodiments are as follows:

[0040] MRS culture medium: 5.0 g / L of yeast powder, 10.0 g / L of beef extract, 10.0 g / L of peptone, 20.0 g / L of glucose, 2.0 g / L of anhydrous sodium acetate, 2.0 g / L of diammonium citrate hydrogen, 2.6 g / L of dipotassium hydrogen phosphate, 0.25 g / L of manganese sulfate monohydrate, 0.5 g / L of magnesium sulfate heptahydrate, 1 mL of Tween-80, pH 6.2 - 6.4.

[0041] BHI culture medium: 10.0 g / L of tryptone, 17.5 g / L of bovine heart infusion powder, 5.0 g / L of sodium chloride, 3.0 g / L of glucose, 2.5 g / L of disodium hydrogen phosphate dodecahydrate, 10.0 g / L of yeast powder, 1.0 g / L of maltose, pH 7.2 - 7.4; when the temperature cools to about 55°C, add 10% sterile fetal bovine serum.

[0042] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content in the mixed solution is 10000 U / mL, streptomycin concentration is 10 mg / mL).

[0043] The strains, cells and animals involved in the following embodiments are as follows:

[0044] SPF-grade BALB / c mice, female, 7 weeks old, weighing 18 - 20 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012 - 0001).

[0045] Gardnerella vaginalis uses Gardnerella vaginalis ATCC 14018, purchased from the GDMCC of the Guangdong Institute of Microbiology.

[0046] Human vaginal epithelial cells VK2 / E6E7 were kindly provided by the Obstetrics and Gynecology Department of the People's Hospital.

[0047] Lactobacillus gasseri QJSWX195M1 and Lactobacillus rhamnosus FXJWS3M4 are preserved at the Food Microbiology Culture Collection Center of Jiangnan University.

[0048] The bacterial solutions involved in the following examples are shown below:

[0049] Lactobacillus rhamnosus CCFM1315 bacterial suspension: Lactobacillus rhamnosus CCFM1315 was inoculated into MRS liquid medium at a 2% inoculum size and incubated at 37°C for 24 hours. The bacterial suspension concentration was then adjusted to ~10. 9 CFU / mL.

[0050] Gardnerella vaginalis suspension: Gardnerella vaginalis strain ATCC 14018 was cultured in BHI medium at 37°C for 24 hours, and the bacterial suspension concentration was adjusted to 10. 10 CFU / mL.

[0051] Example 1: Isolation and Identification of Lactobacillus rhamnosus CCFM1315

[0052] The specific steps are as follows:

[0053] 1. Screening

[0054] The samples were obtained from feces of healthy adults. 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 johnsonii were picked and streaked onto MRS solid medium for purification. Single colonies were picked and transferred to MRS liquid medium for enrichment. The samples were then stored in 30% glycerol to obtain Lactobacillus rhamnosus CCFM1315. The typical colonies of Lactobacillus rhamnosus are round, milky white, and rounded with raised edges.

[0055] 2. Identification

[0056] The genome of strain CCFM1315 was extracted, and the 16S rDNA of the strain was amplified and sequenced (the nucleotide sequence of the 16S rDNA amplified by Shanghai Meiji Biomedical Technology Co., Ltd. is shown in SEQ ID NO.1). The nucleotide sequence of the amplified 16S rDNA of the strain was then compared with the nucleic acid sequence in NCBI. The results showed that the strain was Lactobacillus rhamnosus, and it was named Lactobacillus rhamnosus CCFM1315.

[0057] Example 1: The ability of Lactobacillus to regulate cellular oxidative stress in vitro

[0058] Single colonies were obtained by culturing on MRS solid medium in a water-jacketed incubator at 37°C for 24-48 hours. These single colonies were then inoculated into MRS liquid medium and cultured at 37°C for 12-18 hours to obtain culture medium 1. Culture medium 1 was then inoculated into MRS liquid medium at a 2% (v / v) inoculation rate and cultured at 37°C for 12 hours to obtain seed culture. Seed culture was then inoculated into MRS liquid medium at a 3.5% (v / v) inoculation rate for expansion and cultured at 37°C for 18-24 hours to obtain a concentration of 3.4 × 10⁻⁶. 8 CFU / mL bacterial suspension. The bacterial suspension was homogenized under high pressure (800-1200 MPa, 5 times) to obtain bacterial cell lysate.

[0059] VK2 / E6E7 cells were loaded at 5 × 10 4 Cells were seeded per well in 12-well plates. After culturing for 24 hours, control and treatment groups were set up. The treatment group was supplemented with serum-free medium containing 1.25 μg / mL LPS, while the control group was supplemented with complete medium. After 24 hours, the old medium was discarded, and the cells were washed three times with PBS. The control group was supplemented with 5% MRS medium. The treatment group was supplemented with an equal proportion of bacterial lysate. 2 mL of medium containing bacterial lysate of *Lactobacillus rhamnosus* CCFM1315, *Lactobacillus rhamnosus* FXJWS3M4, *Lactobacillus delbrueckii* DM8909, or *Lactobacillus gasseri* QJSWX195M1 was added to each 12-well plate and cultured for 24 hours. Each sample was tested in triplicate. After co-culture, the culture supernatant was collected, and the Nrf2 content in the vaginal epithelial cell culture supernatant was detected by ELISA. Simultaneously, each well was rapidly washed three times with PBS, and 500 μL of Trizol was added to each well. RNA was extracted by repeated pipetting and collecting. The RNA was then reverse transcribed into cDNA using an RT-PCR reverse transcription kit. Gene expression in VK2 / E6E7 cells was detected using real-time quantitative PCR. -ΔΔCt The formula was used to calculate the expression level of Nrf2 / SOD1 mRNA, with GAPDH as the internal reference. Primers are described in Table 1.

[0060] Table 1 Primers and Sequences

[0061]

[0062] Figure 1(a) The results showed that, compared with the control group (Nrf2 content of 106.10 ng / L), the model group had a lower Nrf2 content of 89.60 ng / L. The Nrf2 content of the bacterial lysates prepared from Lactobacillus delbrueckii DM8909, Lactobacillus rhamnosus CCFM1315, Lactobacillus gasseri QJSWX195M1, and Lactobacillus rhamnosus FXJWS3M4 after intervention was 98.92 ng / L, 141.06 ng / L, 127.73 ng / L, and 80.01 ng / L, respectively. Compared with other groups, the group intervened by Lactobacillus rhamnosus CCFM1315 significantly upregulated the Nrf2 content of vaginal epithelial cells (increasing by 57.43% compared with the model group). The other intervention groups showed the highest increase in Nrf2 content of vaginal epithelial cells compared with the model group, reaching 43.28%.

[0063] Figure 1 (b) The results showed that, with the expression level of Keap1 mRNA in the control group as 1 and the expression level in the model group as 0.50, there was a slight difference compared to the control group. The expression levels of Nrf2 mRNA after intervention with the bacterial lysates prepared from Lactobacillus delbrueckii DM8909, Lactobacillus rhamnosus CCFM1315, Lactobacillus gasseri QJSWX195M1, and Lactobacillus rhamnosus FXJWS3M4 were 1.34, 2.62, 1.92, and 1.47, respectively. Compared with other groups (Lactobacillus delbrueckii DM8909, Lactobacillus rhamnosus FXJWS3M4, p<0.05; Lactobacillus gasseri QJSWX195M1, p<0.01), the Lactobacillus rhamnosus CCFM1315 intervention group significantly upregulated the expression of Nrf2 mRNA (p<0.001). The expression of Keap1 mRNA in vaginal epithelial cells was significantly upregulated (4.24-fold increase compared to the model group), and the Nrf2 content in vaginal epithelial cells in other intervention groups increased by up to 2.84-fold compared to the model group.

[0064] Therefore, Lactobacillus rhamnosus CCFM1315 has the ability to increase Nrf2 levels and upregulate Keap1 expression, showing good potential for regulating antioxidant pathways and improving vaginitis in in vitro experiments.

[0065] Example 2: Application of Lactobacillus rhamnosus CCFM1315 in alleviating vaginitis in mice

[0066] Figure 2 The experimental procedure is as follows:

[0067] Table 2 Experimental Design and Grouping:

[0068] 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 delbrueckii DM8909 Days 1-3 Days 3-7 Days 8-17 Lactobacillus rhamnosus CCFM1315 Days 1-3 Days 3-7 Days 8-17 Lactobacillus gasseri QJSWX195M1 Days 1-3 Days 3-7 Days 8-17

[0069] Mice were randomly divided into 5 groups based on their weight. According to Table 2, all groups of mice were fed normally throughout the experiment.

[0070] Animal model construction: SPF-grade BALB / c mice, female, 7 weeks old, weighing 18-20g, were selected. Mice were injected with estradiol valerate for three consecutive days after the acclimatization period to induce estrus. Gardnerella vaginalis was then used to infect the mice vaginally once daily for 5 consecutive days. The specific infection procedure involved preparing 10... 10 CFU / mL Gardnerella vaginalis suspension, dose 20 μL / mouse. Using a pipette tip, draw 20 μL of the suspension and slowly inject it into the mouse's vagina. Invert the mouse and leave it for 1–2 minutes before placing it in a cage.

[0071] 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 model group received CFU / mL probiotic suspension and underwent only Gardnerella vaginalis infection without subsequent intervention. The intervention group underwent Gardnerella vaginalis infection and then received probiotics for 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 for subsequent intervention.

[0072] 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. For 5 consecutive days (days 3-7), the mice were infected with Gardnerella vaginalis (the specific procedure was to use a pipette tip to draw 20 μL of Gardnerella vaginalis suspension, slowly inject it into the mouse vagina, invert the mouse, leave it for 1-2 minutes, and then place it in a cage). The probiotic intervention group was further intervened with Lactobacillus delbrueckii DM8909, Lactobacillus rhamnosus CCFM1315, and Lactobacillus gasseri QJSWX195M1 for 10 consecutive days (days 8-17). The experimental period was 18 days (days 0-17). At the end of infection (day 7) and the end of intervention (day 17), 50 μL of phosphate buffered solution was aspirated from the vagina of mice using a pipette tip each time. A total of 300 μL of vaginal lavage fluid was collected to determine the load of Gardnerella vaginalis and Lactobacillus in the vagina. Simultaneously, on day 18, all experimental mice were euthanized and vaginal tissue was dissected for subsequent histopathological analysis. The secretion of the antioxidant factor (Nrf2) and the expression of the antioxidant pathway Keap1 / Nrf2 in the vaginal tissue were measured to evaluate the therapeutic effect on bacterial vaginosis in mice. At the same time, the Keap1 mRNA level in colonic tissue was measured to evaluate the intervention effect on intestinal oxidative stress caused by vaginitis in mice.

[0073] Experimental results:

[0074] (1) Expression of the antioxidant pathway Keap1 / Nrf2 in mouse vaginal tissue

[0075] Five groups of mice were sampled at 20 mg 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.).

[0076] Total RNA was extracted from mouse vaginal tissue using the Trizol method, and OD was detected using Nanodrop. 260 / OD 280 The values ​​were determined by reverse transcription. 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 qPCR Master 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 following methods; the primers are shown in Table 3.

[0077] Table 3 Primers and Sequences

[0078]

[0079] Figure 3 (a) The results showed that, compared with the control group (20.48 ng / L), the Nrf2 concentration in the vaginal tissue of the model group mice was significantly reduced (15.95 ng / L, p<0.05). After probiotic intervention, the Nrf2 concentration in the vaginal tissue of mice significantly increased (p<0.0001). Figure 3 (b) The results showed that, with the Keap1 mRNA expression level in the control group as 1, the expression level in the model group was 2.16, significantly higher than that in the control group. The Keap1 mRNA expression levels after intervention with *Lactobacillus delbrueckii* DM8909, *Lactobacillus rhamnosus* CCFM1315, and *Lactobacillus gasseri* QJSWX195M1 were 1.27, 0.57, and 1.13, respectively. Compared with other groups (*Lactobacillus delbrueckii* DM8909, p<0.05; *Lactobacillus gasseri* QJSWX195M1, p<0.01), the *Lactobacillus rhamnosus* CCFM1315 intervention group significantly downregulated Keap1 mRNA expression (p<0.001). *Lactobacillus rhamnosus* CCFM1315 significantly improved the antioxidant capacity of vaginal tissue.

[0080] (2) Keap1 mRNA expression in mouse colon tissue

[0081] Figure 4The results showed that, with the expression level of Keap1 mRNA in the control group as 1, the expression level in the model group was 3.32, significantly higher than that in the control group. The Keap1 mRNA expression levels after intervention with *Lactobacillus delbrueckii* DM8909, *Lactobacillus rhamnosus* CCFM1315, and *Lactobacillus gasseri* QJSWX195M1 were 1.88, 2.13, and 1.90, respectively. After probiotic intervention, the Keap1 mRNA expression level in mouse colon tissue significantly decreased (p<0.0001). *Lactobacillus rhamnosus* CCFM1315 significantly improved the antioxidant capacity of colon tissue.

[0082] (3) Gardnerella vaginalis colonization in mouse vagina

[0083] 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 a soil rapid DNA rotation kit (MP Biomedical, USA) and a QIAQuick 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) consisted of 5 μL of 2×ChamQ Universal SYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL of template DNA (10 ng / μL), 0.5 μL of forward and reverse primers (10 μM each), and 3 μL of double-distilled water. The 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, melting was established by increasing the temperature from 65 °C to 95 °C in 0.5 °C increments every 5 s. 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.

[0084] Table 4. Species-specific primers for real-time quantitative PCR detection of Gardnerella vaginalis.

[0085]

[0086] from Figure 5The results showed that, except for the blank control group, approximately 12.46 lg 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, the colonization level of Gardnerella vaginalis in the CCFM1315 group was significantly reduced to 9.01 lg copies / μL. Compared with the model group, neither Lactobacillus delbrueckii DM8909 nor Lactobacillus gasseri QJSWX195M1 significantly reduced Gardnerella vaginalis colonization (p>0.05).

[0087] (4) Histopathological analysis of mouse vaginal tissue

[0088] At the end of the experiment, mice in all five groups 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 5mm 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).

[0089] HE staining of mouse vaginal tissue can effectively assess the inflammation status of vaginal tissue in different groups of mice. Figure 6 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 interstitial congestion, and a large number of inflammatory cells infiltrating the epithelium and interstitium. After appropriate probiotic intervention for a period of time, the *Lactobacillus delbrueckii* DM8909 group showed less inflammatory cell infiltration than the model group, but significant squamous epithelial hyperplasia was observed. In the other two probiotic intervention groups, *Lactobacillus gasseri* QJSWX195M1 did not significantly improve the recovery of mouse vaginal tissue compared to the model group; compared to the control group, different parts of the vaginal epithelium still showed discontinuity of the vaginal epithelium and inflammatory cell infiltration. *Lactobacillus rhamnosus* CCFM1315 showed the opposite effect to *Lactobacillus gasseri* QJSWX195M1, for example, restoring epithelial continuity, significantly improving interstitial congestion, and showing a small amount of inflammatory cell infiltration. The recovery of vaginal tissue in mice after intervention with *Lactobacillus rhamnosus* CCFM1315 was superior to that in the *Lactobacillus delbrueckii* DM8909 group. In summary, *Lactobacillus rhamnosus* CCFM1315 has a good ability to restore damaged vaginal tissue. The above are merely preferred embodiments of the present invention and are not intended to limit the invention. The 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 invention should also be considered within the protection scope of the invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus CCFM1315, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No:63711.

2. A microbial preparation containing the Lactobacillus rhamnosus CCFM1315 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of *Lactobacillus rhamnosus* CCFM1315 in the microbial preparation is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. A product containing *Lactobacillus rhamnosus* CCFM1315 as described in claim 1 or the microbial preparation as described in claim 2 or 3, characterized in that, The product in question is a pharmaceutical product.

5. The product according to claim 4, characterized in that, The content of *Lactobacillus rhamnosus* CCFM1315 in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

6. The use of the Lactobacillus rhamnosus CCFM1315 of claim 1 or the microbial preparation of claim 2 or 3 in the preparation of a medicine for relieving and / or treating vaginitis.

7. The application according to claim 6, characterized in that, The effects of relieving and / or treating vaginitis include at least one of the following: (1) improving vaginal oxidative stress in mice by activating the Keap1 / Nrf2 pathway in vaginal tissue and increasing the level of Nrf2 in vaginal tissue; (2) improving the antioxidant capacity of colonic tissue by downregulating the expression of Keap1 mRNA in colonic tissue.

8. The application according to claim 6, characterized in that, The dosage forms of the medicine include granules, capsules, tablets, pills, suppositories, or oral liquids.

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