A strain of Lactobacillus paracasei that simultaneously activates immune regulatory and antioxidant pathways to alleviate vaginitis

Abstract

The present invention discloses a strain of Lactobacillus paracasei that simultaneously activates immune regulation and antioxidant pathways to alleviate vaginitis, belonging to the field of microbial technology. The present invention screened and obtained a strain of Lactobacillus paracasei CCFM1316, which has the effect of repairing related symptoms such as immune disorders and oxidative damage caused by vaginitis, specifically embodied in: reducing cellular NF-κB mRNA expression and cell supernatant IL-1β content, improving mouse vaginal immune response, enhancing mouse vaginal antioxidant capacity, improving mouse vaginal pathological manifestations and reducing pathogenic bacteria load. It reduces the mouse vaginal Gardnerella load and improves mouse vaginal pathological manifestations; therefore, Lactobacillus paracasei CCFM1316 has great application prospects in products that improve and repair immune disorders and oxidative damage problems caused by vaginitis.

Description

Technical Field

[0001] The invention relates to a Lactobacillus paracasei strain capable of simultaneously activating immune regulation and antioxidant pathways to alleviate vaginitis, and belongs to the technical field of microorganisms. Background Art

[0002] Gardnerella vaginalis is considered a common core pathogen causing vaginitis, present in over 80% of bacterial vaginosis cases. Invading the vaginal environment, Gardnerella vaginalis adheres to genital epithelial cells, stimulating a local host immune response. Vaginal epithelial cells and immune cells resident in the vaginal tissue are the first to be exposed to the pathogen. Microbial pathogen molecules bind to Toll-like receptors (TLRs), activating the nuclear factor-κB (NF-κB) / myeloid differentiation factor 88 (MyD88) signaling pathway and secreting proinflammatory cytokines such as TNF-α and IL-1β. This triggers an immune response to G. vaginalis in the vaginal mucosa, accompanied by tissue inflammation. Simultaneously, the pathogen damages mitochondria, releasing large amounts of reactive oxygen species (ROS), activating the expression of Kelch-like epichlorohydrin-associated protein-1 (Keap1), and inhibiting the secretion of nuclear factor E2-related factor 2 (Nrf2). This hinders the timely clearance of ROS, leading to oxidative damage.

[0003] Currently, the primary treatment for vaginitis is antibiotics, such as metronidazole and tinidazole. While these medications offer immediate relief from symptoms, statistics show that over 50% of women experience recurrences of vaginitis within a year. This is primarily due to the indiscriminate killing of all vaginal microorganisms by antibiotics, preventing the probiotics from rebuilding the disrupted vaginal microbiome. Once pathogens invade again, there's no adequate bacterial environment to defend against them. Furthermore, antibiotics have no ability to repair a damaged vaginal environment. Damaged vaginal barriers attacked by inflammatory factors and reactive oxygen species can only be restored through the body's own metabolism.

[0004] From a microbiome perspective, taking probiotics has advantages in alleviating vaginitis. Studies have found that oral probiotic therapy can mildly improve vaginal microecological imbalances in pregnant women ("Intervention of Oral Probiotics on Intestinal and Vaginal Colonization of Escherichia coli K1 and Group B Streptococci in Pregnant Women and Its Effect on Vaginal Microecology," Southern Medical University, 2019). However, there are very limited domestic probiotic products currently available on the market for the treatment of bacterial vaginosis. In 1989, the Institute of Microecology at Dalian Medical University isolated and screened a Lactobacillus DM8909 strain from the vaginal secretions of healthy women. Experimental data ("Study on the Inhibition of Vaginal Infection in Mice by Lactobacillus DM8909," Chinese Journal of Microecology, 2003; "Study on the Inhibitory Effect of Lactobacillus DM8909 on the Adhesion of Escherichia coli to Vaginal Epithelial Cells," Chinese Journal of Microecology, 1997; "Preliminary Study on the Adhesion of Lactobacillus DM8909 to Vaginal Epithelial Cells," Chinese Journal of Microecology, 1993) and clinical data ("Phase II Clinical Trial of Lactobacillus delbrueckii DM8909 for the Treatment of Bacterial Vaginosis," Chinese Journal of Microecology, 2001) demonstrated that Lactobacillus delbrueckii DM8909 possesses beneficial probiotic properties, such as a certain degree of adhesion to vaginal epithelial cells. It also inhibits the growth and reproduction of harmful pathogens, regulates the balance of vaginal flora, and is effective in treating bacterial vaginosis. However, specific reports on the effects of this strain on the immune disorders and oxidative damage that cause vaginitis have not been published.

[0005] Existing partial patent application relates to the ability (CN116426435A and CN116179413A) of Lactobacillus paracasei to inhibit the growth of pathogenic bacteria, and the bacterial strain protected therein has the ability of lowering vaginal tissue inflammatory factors in vivo, but how to lower the immune response is not clear, and there is no corresponding research on the oxidative damage caused by vaginitis. Therefore, there is no Lactobacillus paracasei that illustrates the mechanism, clearly can be used to alleviate bacterial vaginosis. Summary of the Invention

[0006] The present invention provides a strain of Lactobacillus paracasei that can alleviate bacterial vaginosis and improve the immune disorder and oxidative damage caused by vaginitis; the Lactobacillus paracasei CCFM1316 has a good ability to inhibit the expression of NF-κB mRNA in vaginal epithelial cells and the content of IL-1β in cell supernatant in vitro, and has the potential to alleviate bacterial vaginosis. SPF-grade BALB / c female mice, 7 weeks old, weighing 18-20g were selected. After the adaptation period, the mice were injected with estradiol valerate for three consecutive days to induce estrus. The mice were continuously infected with Gardnerella vaginalis once a day for 5 days. The specific operation method of the infection is to prepare 10 10CFU / mL vaginal Gardnerella suspension, the dose is 20μL / mouse. Use the pipette tip to draw 20μL of bacterial suspension and slowly inject it into the mouse vagina. The mouse is inverted and stays for 1-2 minutes and then placed in a cage. The mice with bacterial vaginosis model were divided into model group and intervention group. Among them, the intervention group was gavaged with 100μL of 10 9 CFU / mL of probiotic suspension, the model group was simply infected with vaginal Gardnerella and did not receive subsequent intervention, the intervention group was infected with vaginal Gardnerella and then received probiotics for subsequent intervention; in addition, a blank control group was set up, in which the blank control group was not infected with vaginal Gardnerella and did not receive subsequent probiotic intervention; during the experiment, the therapeutic effect of bacterial vaginosis in mice was evaluated by analyzing the vaginal colonization of vaginal Gardnerella, vaginal epithelial cell shedding, inflammatory factor (IL-1β) secretion, immune pathway (NF-κB / MyD88) expression, antioxidant pathway (Keap1) expression and tissue pathology.

[0007] The present invention provides a Lacticaseibacillus paracasei CCFM1316; the Lacticaseibacillus paracasei CCFM1316 was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 4, 2023, with a deposit number of GDMCC No: 63712.

[0008] The invention provides a microbial agent, which contains Lacticaseibacillus paracasei CCFM1316 or its fermentation liquid, or its lysate.

[0009] In one embodiment of the present invention, the microbial agent is a liquid preparation, a powder or a granular preparation.

[0010] In one embodiment of the present invention, the microbial agent is Lacticaseibacillus paracasei CCFM1316 freeze-dried powder.

[0011] In one embodiment of the present invention, in the above-mentioned microbial agent, the addition amount of the Lacticaseibacillus paracasei CCFM1316 is at least: 10 6 CFU / mL or 10 6 CFU / g.

[0012] The invention provides a product, which contains the Lactobacillus paracasei CCFM1316 or the microbial agent.

[0013] In one embodiment of the present invention, in the above product, the addition amount of the Lactobacillus paracasei CCFM1316 is at least: 10 6 CFU / mL or 10 6 CFU / g.

[0014] In one embodiment of the present invention, the product is a medicine or a sanitary product.

[0015] In one embodiment of the present invention, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0016] In one embodiment of the present invention, the medicine comprises the above composition and a pharmaceutically acceptable carrier.

[0017] In one embodiment of the present invention, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0018] In one embodiment of the present invention, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0019] In one embodiment of the present invention, the medicine includes oral tablets and capsules with enteric coating, oral liquid; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0020] The present invention also provides the use of the above-mentioned Lactobacillus paracasei CCFM1316 or the above-mentioned microbial agent in preparing products for alleviating and / or treating bacterial vaginosis or anti-aging.

[0021] In one embodiment of the present invention, in the above product, the addition amount of the Lactobacillus paracasei CCFM1316 is at least: 10 6 CFU / mL or 10 6 CFU / g.

[0022] In one embodiment of the present invention, the product is a medicine or a sanitary product.

[0023] In one embodiment of the present invention, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0024] In one embodiment of the present invention, the medicine comprises the above composition and a pharmaceutically acceptable carrier.

[0025] In one embodiment of the present invention, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0026] In one embodiment of the present invention, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0027] In one embodiment of the present invention, the medicine includes oral tablets and capsules with enteric coating, oral liquid; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0028] The present invention also provides the use of the above-mentioned Lactobacillus paracasei CCFM1316 or the above-mentioned microbial agent in preparing a microbial preparation having any of the following uses:

[0029] (1) Reduce cellular NF-κB mRNA expression and IL-1β content in cell supernatant;

[0030] (2) reduce vaginal Gardnerella load in mice;

[0031] (3) Improve vaginal pathology in mice;

[0032] (4) Improved vaginal immune response in mice: reduced NF-κB / MyD88 mRNA expression and decreased IL-1β content in vaginal tissue;

[0033] (5) Enhance the antioxidant capacity of mouse vagina: reduce Keap1 mRNA expression.

[0034] (6) Improve vaginal pathological manifestations and reduce pathogenic bacteria load in mice; Reduce vaginal Gardnerella load in mice;

[0035] (7) Improve vaginal pathological manifestations in mice.

[0036] Beneficial effects

[0037] The present invention screened and obtained a strain of Lacticaseibacillus paracasei CCFM1316, which has the effect of repairing the immune disorder, oxidative damage and other related symptoms caused by vaginitis, which is specifically reflected in:

[0038] (1) Reduce cellular NF-κB mRNA expression and IL-1β content in cell supernatant;

[0039] (2) Improved vaginal immune response in mice: reduced NF-κB / MyD88 mRNA expression and decreased IL-1β content in vaginal tissue;

[0040] (3) Enhanced vaginal antioxidant capacity in mice: reduced Keap1 mRNA expression;

[0041] (4) Improve vaginal pathological manifestations and reduce pathogenic bacteria load in mice; Reduce vaginal Gardnerella load in mice;

[0042] (5) Improve vaginal pathology in mice;

[0043] Therefore, Lacticaseibacillus paracasei CCFM1316 has great application prospects in products that improve and repair the immune disorders and oxidative damage problems caused by vaginitis.

[0044] Biomaterial Deposit

[0045] A strain of Lacticaseibacillus paracasei CCFM1316, taxonomically named Lacticaseibacillus paracasei, was deposited in the Guangdong Provincial Microbial Culture Collection on August 4, 2023, with the deposit number GDMCC No: 63712. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Effects of different bacterial lysates on IL-1β content and NF-κB mRNA expression in the culture supernatant of vaginal epithelial cells VK2 / E6E7; compared with the model group, **p<0.01, ***p<0.001.

[0047] Figure 2 : Flowchart of animal experiment design plan.

[0048] Figure 3 : Effect of Lactobacillus paracasei on the concentration of vaginal proinflammatory factor IL-1β; compared with the model group, *p<0.05.

[0049] Figure 4 : Effects of Lactobacillus paracasei on vaginal inflammation pathways; a is the effect on NF-κB mRNA expression; b is the effect on MyD88 mRNA expression; compared with the model group, ***p<0.001, ****p<0.0001.

[0050] Figure 5 : Effect of Lactobacillus paracasei on Keap1 mRNA expression; **p<0.01, ***p<0.001, ****p<0.0001.

[0051] Figure 6 : Diagram of vaginal colonization of Gardnerella vaginalis; Gardnerella vaginalis load in the vagina of BV mice on days 7 and 17; a, b represent significant differences between different groups on day 17 (p<0.05).

[0052] Figure 7 : Figure 3 histopathological evaluation of mouse vagina. DETAILED DESCRIPTION

[0053] The vaginal epithelial cells VK2 / E6E7 used in the following examples were kindly provided by the Department of Obstetrics and Gynecology, Renmin Hospital. Gardnerella vaginalis ATCC 14018 was purchased from the Guangdong Provincial Microbiology Institute Culture Collection Center (GDMCC).

[0054] The Lactobacillus gasseri QJSWX195M1 and Lactobacillus paracasei FFJLY55L1 involved in the following examples are deposited in the Food Microorganism Culture Collection Center of Jiangnan University.

[0055] The culture medium involved in the following examples is as follows:

[0056] MRS medium: yeast powder 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 hydrogen 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.

[0057] BHI medium: 10.0 g / L tryptone, 17.5 g / L beef heart extract powder, 5.0 g / L sodium chloride, 3.0 g / L glucose, 2.5 g / L disodium hydrogen phosphate dodecahydrate, 10.0 g / L yeast extract, 1.0 g / L maltose, pH 7.2-7.4; after cooling to approximately 55°C, add 10% sterile fetal bovine serum.

[0058] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin.

[0059] and streptomycin mixed solution (penicillin content in the mixed solution is 10000U / mL, streptomycin concentration is 10mg / mL).

[0060] Lactobacillus paracasei CCFM1316 bacterial suspension: Lactobacillus paracasei CCFM1316 was inoculated in MRS liquid medium at a 2% inoculum volume, and cultured in a 37°C incubator for 24 h to adjust the bacterial suspension concentration to 1×10 9 CFU / mL.

[0061] Vaginal Gardnerella suspension: Vaginal Gardnerella strain ATCC 14018 was cultured in BHI medium at 37°C for 24 h, and the bacterial suspension concentration was adjusted to 10 10 CFU / mL.

[0062] The detection methods involved in the following embodiments are as follows:

[0063] Detection of NF-κB mRNA expression in VK2 / E6E7:

[0064] 2 mL of culture medium containing bacterial lysates of Lactobacillus paracasei CCFM1316, Lactobacillus paracasei FFJLY55L1, Lactobacillus delbrueckii DM8909 or Lactobacillus gasseri QJSWX195M1 was respectively added to a 12-well plate and cultured for 24 hours, with three parallels for each sample. After the co-culture was completed, the culture supernatant was collected and the IL-1β content in the culture supernatant of vaginal epithelial cells was detected by ELISA. At the same time, each well was quickly washed 3 times with PBS, 500 μL Trizol was added to each well, and RNA was repeatedly pipetted and collected to extract RNA, and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in VK2 / E6E7 cells was detected by real-time fluorescence quantification, and 2 -ΔΔCt The expression of NF-κB mRNA was calculated using the formula, where GAPDH was the internal reference and the primers are described in Table 1.

[0065] Table 1 Primers and sequences

[0066]

[0067] Secretion of the immunomodulatory factor IL-1β in mouse vaginal tissue

[0068] For cytokine analysis, 20 mg of mouse vaginal tissue was homogenized with 180 μL of pre-chilled PBS. The sample was centrifuged at 3000 rpm for 15 min at 4°C, and the vaginal tissue supernatant was used to determine IL-1β concentration according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.).

[0069] Expression of NF-κB / MyD88 mRNA in mouse vaginal tissue

[0070] Total RNA was extracted from mouse vaginal tissue using Trizol method, and OD was detected by Nanodrop. 260 / OD 280 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit and used in the fluorescent quantitative PCR reaction system with ChamQ Universal SYBR qPCR Master Mix. GAPDH was used as the internal reference gene based on 2-ΔΔCt Methods The expression level of target genes was calculated. The primers are shown in Table 2.

[0071] Table 2 Primers and sequences

[0072]

[0073] Keap1 mRNA expression in mouse vaginal tissue

[0074] Total RNA was extracted from mouse vaginal tissue using Trizol method, and OD was detected by Nanodrop. 260 / OD 280 1 μg of total RNA was reverse transcribed into 20 μL of cDNA using a reverse transcription kit and used in the fluorescent quantitative PCR reaction system with ChamQ Universal SYBR qPCR Master Mix. GAPDH was used as the internal reference gene, based on 2 -ΔΔCt Methods The expression level of target genes was calculated. The primers are shown in Table 3.

[0075] Table 3 Primers and sequences

[0076]

[0077]

[0078] Gardnerella vaginalis colonization load in mouse vagina

[0079] Vaginal lavage fluid samples were obtained by pipetting a phosphate-buffered solution (PBS) into the mouse vagina using a pipette tip and then aspirating back and forth to obtain 300 μL of fluid. Gardnerella vaginalis and Lactobacillus gasseri were then counted using qPCR. The entire experimental period lasted 17 days, with two samplings performed. The first sample was collected on day 7 (the first day after infection) to test for G. vaginalis colonization. The second sample was collected after the intervention (day 17) to quantify G. vaginalis and Lactobacillus gasseri. First, DNA was extracted from vaginal lavage fluid using the Soil Rapid DNA Spin Kit (MP Biomedical, USA) and the QIAQuick Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions. Subsequently, G. vaginalis and L. gasseri were quantified using qPCR. Primers were selected based on the bacterial 16S rRNA sequence. The reaction mixture (10 μL) included 5 μL of 2× ChamQ Universal SYBR qPCR Master Mix (Nanjing Novozyme 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 as follows: initial denaturation at 95°C for 30 s; followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Another step, 95°C for 10 s, was followed by an increase from 65°C to 95°C in 0.5°C increments every 5 s 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 examined in triplicate.

[0080] Table 4: Species-specific primers for real-time quantitative PCR detection of Gardnerella vaginalis

[0081]

[0082] Mouse vaginal histopathological analysis

[0083] At the end of the experiment, mice were sacrificed and their vaginas were excised. A portion of vaginal tissue was used for histopathological examination. Vaginal tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into 5-mm thick sections. Hematoxylin and eosin (H&E) staining was performed. Vaginal tissue samples were examined at 40x magnification using a pathology slide scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary).

[0084] Example 1: Isolation and Identification Method of Lacticaseibacillus paracasei CCFM1316

[0085] The specific steps are as follows:

[0086] 1. Screening

[0087] The samples were derived from feces of healthy adults. After pretreatment, the samples were stored in a -80°C refrigerator in 30% glycerol. After thawing, they were mixed and 0.5 mL of the sample was added to 4.5 mL of normal saline. The samples were gradiently diluted with 9 g / L normal saline. Appropriate gradient dilutions were selected and spread on MRS solid culture medium. The samples were cultured at 37°C for 48 hours. Typical colonies of Lactobacillus paracasei were picked and streaked on MRS solid culture medium for purification. Single colonies were picked and transferred to MRS liquid culture medium for enrichment and preserved in 30% glycerol to obtain Lactobacillus paracasei CCFM1316. Among them, the typical colonies of Lactobacillus paracasei are round, milky white, and smooth and raised.

[0088] 2. Identification

[0089] The genome of strain CCFM1316 was extracted, and the 16S rDNA of the strain was amplified and sequenced (performed by Shanghai Meiji Biomedical Technology Co., Ltd., and the nucleotide sequence of the 16S rDNA amplified from CCFM1316 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 gasseri, and was named Lacticaseibacillus paracasei CCFM1316.

[0090] Example 2: Ability of Lactobacillus to regulate vaginitis immunity in vitro

[0091] The specific steps are as follows:

[0092] (1) Preparation of bacterial suspension

[0093] Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1316, Lactobacillus gasseri QJSWX195M1, and Lactobacillus paracasei FFJLY55L1 are inoculated into MRS solid culture medium respectively, and cultured in a water-tight constant temperature incubator at 37°C for 24-48 hours to obtain single colonies; the single colonies are picked and inoculated into MRS liquid culture medium, and cultured at 37°C for 12-18 hours to obtain culture solution; the culture solution is inoculated into the MRS liquid culture medium at an inoculum amount of 2% (v / v), and cultured at 37°C for 12 hours to obtain seed solution;

[0094] The seed solution was inoculated into MRS liquid medium at an inoculum volume of 3-5% (v / v) and cultured at 37°C for 18-24 h to obtain a concentration of 3.4×10 8 CFU / mL of bacterial solution; the bacterial solution was homogenized in a high-pressure homogenizer (800-1200 MPa, 5 times) to obtain bacterial lysate.

[0095] Vaginal epithelial cells VK2 / E6E7 were cultured at a density of 2.5×10 4 12-well plates were inoculated with 2 mL of culture medium per well. After culturing the cells for 24 h, a control group and a treatment group were set up. The treatment group was supplemented with serum-free medium at a concentration of 1.25 μg / mL of LPS, while the control group was supplemented with complete medium. After 24 h, the old medium was discarded and the cells were rinsed three times with PBS. The control group was supplemented with 5% MRS medium; the treatment group was supplemented with 5% bacterial lysate. The expression of NF-κB mRNA was calculated. Figure 1 shown.

[0096] The results show:

[0097] (1) By Figure 1 As can be seen from a in the figure, compared with the IL-1β content of 5.24 ng / L in the control group, the IL-1β content in the model group was 6.92 ng / L, and the IL-1β content was significantly increased. The IL-1β contents of the bacterial lysates prepared from Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1316, Lactobacillus gasseri QJSWX195M1, and Lactobacillus paracasei FFJLY55L1 after intervention were 6.05 ng / L, 4.71 ng / L, 6.65 ng / L, and 5.78 ng / L, respectively. It can be seen that compared with other groups, the group intervened by Lactobacillus paracasei CCFM1316 significantly decreased the IL-1β content of vaginal epithelial cells (decreased by 31.94% compared with the model group), and the IL-1β content of vaginal epithelial cells in other intervention groups decreased by the highest 16.47% compared with the model group.

[0098] (2) By Figure 1 As can be seen from b, the expression level of NF-κB mRNA in the control group was 1, and the expression level in the model group was 2.18, which was significantly increased compared with the control group. The NF-κB mRNA expression levels of the bacterial lysates prepared from Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1316, Lactobacillus gasseri QJSWX195M1, and Lactobacillus paracasei FFJLY55L1 after intervention were 1.00, 0.87, 1.72, and 1.29, respectively; it can be seen that compared with other groups (Lactobacillus gasseri QJSWX195M1), the Lactobacillus paracasei CCFM1316 intervention group significantly reduced the expression of NF-κB mRNA (reduced by 60.09% compared with the model group).

[0099] Activation of the NF-κB signaling pathway leads to the secretion of pro-inflammatory cytokines, triggering an immune response in the vaginal mucosa and accompanied by tissue inflammation. Cell-based experiments have shown that while Lactobacillus paracasei CCFM1316 downregulates NF-κB expression, it also reduces IL-1β levels.

[0100] Example 3: Application of Lactobacillus paracasei CCFM1316 in Alleviating Vaginitis in Mice

[0101] The specific steps are as follows:

[0102] (1) Preparation of bacterial suspension

[0103] Lactobacillus was inoculated into MRS solid culture medium respectively, and cultured in a water-tight constant temperature incubator at 37°C for 24-48 hours to obtain a single colony; a single colony was picked and inoculated into MRS liquid culture medium, and cultured at 37°C for 12-18 hours to obtain a culture solution;

[0104] The culture solution was inoculated into MRS liquid medium at an inoculum volume of 2% (v / v), and cultured at 37°C for 12 hours to obtain seed solution; the seed solution was inoculated into MRS liquid medium at a volume of 3-5% (v / v) for expansion, and cultured at 37°C for 18-24 hours to obtain a concentration of 10 9 CFU / mL of bacterial solution.

[0105] (2) Animal experiments. For detailed experimental procedures, see Figure 2 And Table 5:

[0106] Table 5 Experimental plan and grouping:

[0107] Grouping Inducing estrus Induction time of Gardnerella 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 paracasei CCFM1316 Days 1-3 Days 3-7 Days 8-17 Lactobacillus gasseri QJSWX195M1 Days 1-3 Days 3-7 Days 8-17

[0108] SPF-grade BALB / c female mice, 7 weeks old, weighing 18-20 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001). The mice were randomly divided into 5 groups according to their body weight. Refer to Table 5. All groups of mice were kept normally throughout the experiment. Both the bacterial vaginosis model group and the intervention group were required to undergo 3 consecutive days (1-3 days) of subcutaneous injection of 100 μL estradiol valerate solution (0.5 mg estradiol valerate dissolved in 100 μL filter-sterilized sesame oil) in the neck to induce estrus. Vaginal infection was carried out for 5 consecutive days (3-7 days) (the specific operation method of infection was to use a pipette to absorb 20 μL of vaginal Gardnerella suspension, slowly inject it into the mouse vagina, and then put the mouse upside down, stay for 1-2 minutes, and then put it in a cage). The experimental period was 18 days (days 1 to 18):

[0109] Estrus induction: 100 μL of estradiol valerate solution (0.5 mg of estradiol valerate dissolved in 100 μL of filter-sterilized sesame oil) was injected subcutaneously into the neck on day 1-3 to induce estrus.

[0110] Infection experiment (modeling period): On the third day, the blank control group mice were inoculated with 20 μL PBS in the vagina, and the other mice were inoculated with 20 μL of viable bacteria in the vagina. 10Vaginal Gardnerella suspension containing 100 CFU / mL. After inoculation, invert the mouse for 1-2 minutes to prevent bacterial outflow. Repeat this for 5 consecutive days until the end of vaginal Gardnerella inoculation on the 7th day.

[0111] Intervention experiment: Starting from day 8, the details are as follows:

[0112] Blank control group: 100 μL PBS was gavaged once a day;

[0113] Model group mice: gavage with 100 μL PBS once a day;

[0114] Lactobacillus delbrueckii DM8909 group: mice were gavaged once a day with 100 μL, 10 9 CFU / mL Lactobacillus delbrueckii DM8909 bacterial suspension;

[0115] Lactobacillus paracasei CCFM1316 group: mice were gavaged once a day with 100 μL, 10 9 CFU / mL Lactobacillus paracasei CCFM1316 bacterial suspension;

[0116] Lactobacillus gasseri QJSWX195M1 group: mice were gavaged once a day with 100 μL, 10 9 CFU / mL Lactobacillus gasseri QJSWX195M1 bacterial suspension;

[0117] At the end of infection (day 7) and intervention (day 17), 50 μL of phosphate buffer solution was sucked out of the mouse vagina for sampling with a pipette tip, and finally 300 μL of vaginal lavage fluid was collected to measure the vaginal Gardnerella and Lactobacillus load.

[0118] At the same time, on the 18th day, all experimental mice were killed and the vaginal tissue was peeled off for subsequent experimental histopathological analysis. The secretion of inflammatory factors (IL-1β), the expression of immune pathways (NF-κB / MyD88), and the expression of antioxidant pathways (Keap1) in the vaginal tissue were detected to evaluate the therapeutic effect of bacterial vaginosis in mice and to evaluate the intervention effect on the immune disorders and oxidative stress problems caused by vaginitis in mice.

[0119] (3) Experimental results:

[0120] 1) Secretion of the immunomodulatory factor IL-1β in mouse vaginal tissue

[0121] The vaginal tissue supernatant was collected and the IL-1β concentration was determined according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). Figure 3 shown.

[0122] The results show:

[0123] Compared to the control group (24.31 ng / L), the vaginal tissue of the model group mice secreted a large amount of IL-1β, at a concentration of 42.73 ng / L. After probiotic intervention, Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri QJSWX195M1 did not significantly regulate proinflammatory factors compared to the model group. However, Lactobacillus paracasei CCFM1316 significantly reduced the expression of the proinflammatory cytokine IL-1β in vaginal tissue (35.59 ng / L) compared to the other groups, with an inhibition rate of 16.71%.

[0124] 2) NF-κB / MyD88 mRNA expression in mouse vaginal tissue

[0125] The total RNA of mouse vaginal tissue was extracted by Trizol method, GAPDH was used as the internal reference gene, and the results were analyzed based on 2 -ΔΔCt The expression level of target gene was calculated by this method. The results are shown in Figure 4 shown.

[0126] The results show:

[0127] Depend on Figure 4 As can be seen from a in the figure, the expression level of NF-κB mRNA in the control group was 1, and the expression level in the model group was 2.44, which was significantly increased compared with the control group. The NF-κB mRNA expression levels after intervention with Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1316, and Lactobacillus gasseri QJSWX195M1 were 1.05, 0.89, and 1.79, respectively. Compared with other groups (Lactobacillus gasseri QJSWX195M1), the Lactobacillus paracasei CCFM1316 intervention group significantly reduced the expression of NF-κB mRNA (reduced by 63.52% compared with the model group).

[0128] Depend on Figure 4 As shown in Figure b, the MyD88 mRNA expression level in the control group was 1, and the expression level in the model group was 3.13, which was significantly increased compared to the control group. The MyD88 mRNA expression levels after intervention with Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1316, and Lactobacillus gasseri QJSWX195M1 were 0.94, 0.51, and 3.53, respectively. Compared with the other groups (Lactobacillus gasseri QJSWX195M1), the Lactobacillus paracasei CCFM1316 intervention group significantly reduced MyD88 mRNA expression (compared to the model group, it decreased by 83.71%). Lactobacillus paracasei CCFM1316 can significantly reduce the expression of NF-κB / MyD88 mRNA levels in vaginal tissue.

[0129] 3) Keap1 mRNA expression in mouse vaginal tissue

[0130] The total RNA of mouse vaginal tissue was extracted by Trizol method, GAPDH was used as the internal reference gene, and the results were analyzed based on 2 -ΔΔCt The expression level of target gene was calculated by this method; the results were as follows Figure 5 shown.

[0131] The results showed that the expression level of Keap1 mRNA in the control group was 1, and the expression level in the model group was 2.16, which was significantly increased compared with the control group. The Keap1 mRNA expression levels after intervention with Lactobacillus delbrueckii DM8909, Lactobacillus paracasei CCFM1316, and Lactobacillus gasseri QJSWX195M1 were 1.27, 0.37, and 1.12, respectively. Compared with other groups (Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri QJSWX195M1), the Lactobacillus paracasei CCFM1316 intervention group most significantly reduced the expression of Keap1 mRNA (reduced by 82.87% compared with the model group).

[0132] (4) Colonization load of Gardnerella vaginalis in mouse vagina

[0133] The sampling method was to use a pipette to suck a certain amount of phosphate buffer solution and blow it back and forth into the mouse vagina to obtain a 300μL vaginal lavage sample, and the vaginal Gardnerella and Lactobacillus were counted using qPCR. The entire experimental period was 17 days, during which two samplings were performed. The first sampling was on the 7th day (the first day after the infection) as a colonization test for vaginal Gardnerella, and the second sampling colonization test was after the intervention (17th day) to quantify the vaginal Gardnerella and Lactobacillus load. The results are as follows Figure 6 shown.

[0134] The results show:

[0135] In all groups except the blank control group, approximately 12.44 lg copies / μL of vaginal Gardnerella could be detected in the mice on the 7th day, indicating that vaginal Gardnerella was successfully colonized.

[0136] In the blank control group, no vaginal Gardnerella was detected throughout the experiment. After probiotic intervention (Day 17), vaginal Gardnerella colonization in the CCFM1316 group was significantly reduced to 9.83 lg copies / μL. Compared with the model group, neither Lactobacillus delbrueckii DM8909 (10.72 lg copies / μL) nor Lactobacillus gasseri QJSWX195M1 (10.78 lg copies / μL) significantly reduced vaginal Gardnerella colonization (p>0.05).

[0137] (5) Mouse vaginal histopathological analysis

[0138] HE staining of mouse vaginal tissue can effectively evaluate the inflammation of the vaginal tissue of mice in each group.

[0139] The results are as follows Figure 7 shown.

[0140] The results show:

[0141] The vaginal epithelium in the blank control group was smooth and continuous, with intact tissue structure and no significant inflammatory cell infiltration. In the model group, the vaginal epithelium showed poor epithelial continuity, with surface cell erosion and pore formation, submucosal interstitial congestion, and numerous inflammatory cells infiltrating both the epithelium and interstitium. After a period of appropriate probiotic intervention, the inflammatory cell infiltration in the Lactobacillus delbrueckii DM8909 group was less than that in the model group, but significant squamous epithelial hyperplasia was present. Of the other two probiotic intervention groups, Lactobacillus gasseri QJSWX195M1 did not significantly improve vaginal tissue recovery compared to the model group. Compared to the control group, discontinuity and inflammatory cell infiltration persisted in various locations within the vaginal epithelium. Lactobacillus paracasei CCFM1316, however, exhibited the opposite effects of Lactobacillus gasseri QJSWX195M1, with restoration of epithelial continuity, significant improvement in interstitial congestion, and minimal inflammatory cell infiltration. Vaginal tissue recovery in mice treated with Lactobacillus paracasei CCFM1316 was superior to that in the Lactobacillus delbrueckii DM8909 group.

[0142] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei )CCFM1316 was deposited in Guangdong Provincial Microbiological Culture Collection on August 4, 2023, with the deposit number GDMCC No: 63712.

2. A microbial agent, characterized in that: The microbial agent contains the Lactobacillus paracasei CCFM1316 according to claim 1 or its fermentation liquid or lysate.

3. A product, characterized in that The product contains the Lactobacillus paracasei CCFM1316 according to claim 1 or the microbial agent according to claim 2, and the product is a medicine or a sanitary product.

4. The product according to claim 3, characterized in that The sanitary product is a sanitary wet wipe.

5. The product according to claim 3, characterized in that The sanitary product is a sanitary napkin or a sanitary pad.

6. The product according to claim 3, characterized in that The product is a vaginal wash.

7. The product according to claim 3, characterized in that The product described is an antibacterial wash for women.

8. The product according to claim 3, characterized in that The dosage form of the medicine is granules, capsules or tablets.

9. The product according to claim 3, characterized in that The dosage form of the medicine is pill.

10. The product according to claim 3, characterized in that The dosage form of the medicine is suppository.

11. Use of the Lactobacillus paracasei CCFM1316 according to claim 1 or the microbial agent according to claim 2 in preparing a product for alleviating and / or treating bacterial vaginosis, characterized in that: The product is a medicine or a sanitary product.

12. The use according to claim 11, wherein the sanitary product is a sanitary wet wipe.

13. The use according to claim 11, wherein the sanitary product is a sanitary napkin or a panty liner.

14. The use according to claim 11, wherein the product is a vaginal wash.

15. The use according to claim 11, wherein the product is an antibacterial wash for women.

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