Lactobacillus crispatus strain based on host immune regulation to alleviate various vaginitis

Abstract

The application discloses a lactobacillus crispatus for relieving various vaginitis based on host immune regulation, and belongs to the technical field of microorganisms. The application provides a lactobacillus crispatus CCFM1298, which is preserved in the Guangdong Microbial Culture Collection Center on December 9, 2022, and has a preservation number of GDMCC No: 62275. The lactobacillus crispatus CCFM1298 targets the vaginal immune system, down-regulates pro-inflammatory factors IL-1beta and TNF-alpha, and up-regulates the expression of anti-inflammatory factor IL-10, is suitable for relieving various vaginal inflammations and pathological improvement, and significantly reduces the shedding of vaginal epithelial cells. The lactobacillus crispatus is derived from the vagina of a healthy female, is a probiotic strain suitable for human vaginal application, helps to relieve and improve various vaginal infections of females, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a lactobacillus crispatus based on host immune regulation to alleviate various vaginitis, belonging to the field of microbial technology. BACKGROUND

[0002] In healthy women, the vaginal flora is approximately divided into five large clusters, respectively CST I (Lactobacillus crispatus is dominant), CST II (Lactobacillus gasseri is dominant), CST III (Lactobacillus iners is dominant), CST IV (Lactobacillus and bacteria associated with bacterial vaginosis), and CST V (Lactobacillus jensenii is dominant). These lactobacilli present in the vagina metabolize the glycogen secreted by the vaginal epithelium to produce organic acids, maintaining the normal acidic environment of the vagina (pH <4.5). When lactobacilli are lacking in the vaginal flora, and pathogenic bacteria grow excessively, it leads to symptomatic or asymptomatic infection. Among them, bacterial vaginosis (BV) and vulvovaginal candidiasis (or candidal vaginitis, VVC) are the most common vaginal infections in women of childbearing age.

[0003] BV is a vaginal infection caused by a decrease or absence of lactobacilli that normally produce hydrogen peroxide in the vagina, and by the predominance of facultative and anaerobic bacteria. The main clinical manifestations are increased vaginal discharge, fishy odor, and mild vulvar itching or burning. The discharge is grayish, homogeneous, and thin. The initial cure rate of BV is 70%-90%, and the recurrence rate 3 months after treatment can reach 40%, and the recurrence rate 12 months after treatment can reach 60%. VVC affects millions of women every year, and 75% of women have experienced VVC at least once during their reproductive years. The most common clinical manifestations are vulvar itching and burning, accompanied by vaginal pain and irritation, leading to dysuria. Erythema, edema, and fissuring of the vulva and vagina are also common. At the same time, BV and / or VVC are associated with many complications, including premature birth, pelvic inflammation, infertility, ectopic pregnancy, pelvic abscess, spontaneous abortion, menstrual disorders, increased susceptibility to HIV infection, and other chronic health problems. Given the prevalence of BV and VVC and related complications, BV and VVC have been recognized as important global public health problems.

[0004] In women with BV, Gardnerella vaginalis and Prevotella vaginalis are the most common pathogens, while Candida species are most frequently associated with VVC, with Candida albicans, Candida glabrata, and Candida tropicalis being the most prevalent. These opportunistic pathogens change from a symbiotic state to a pathogenic state depending on changes in host conditions. The basic commonalities of these pathogens include adhesion and biofilm formation, invasion of host cells, secretion of virulence factors, acquisition of host nutrients, and evasion of host defense mechanisms. Antibiotics are currently the first-line treatment for BV and VVC, but their use is associated with high failure and recurrence rates. This may be due to increased antibiotic resistance in pathogens, poor antibiotic permeability to pathogen biofilms, failure to restore the vaginal acidic environment, and the disruption of the normal vaginal microecology.

[0005] Over the past few decades, probiotics have demonstrated good antibacterial activity, with mechanisms including inhibiting pathogenic bacteria adhesion sites; inhibiting adhesion through copolymerization; and secretion of antibacterial molecules. Currently, there are very few domestically produced probiotic products on the market for the adjunctive relief / treatment of vaginitis. These mainly include a strain of *Lactobacillus delbrueckii* DM8909 screened from vaginal secretions of healthy women by Professor Kang Bai of Dalian Medical University (Microbiological Study of *Lactobacillus delbrueckii* DM8909 strain [J]. Chinese Journal of Microecology, 2001) and *Enterococcus* produced by Xi'an Zhenghao Biopharmaceutical Co., Ltd. However, neither of these strains were screened from the dominant flora in the vagina of healthy women of childbearing age in my country, and the discussion of the therapeutic effect of this strain on vaginitis is limited to competitive exclusion and the use of a single antibacterial substance (Kang Bai, Lü Hu, Fu Xiaoli, et al. Study on the mechanism of action of DM8909 strain in treating dysbiosis vaginitis [J]. Chinese Journal of Microecology, 2001). Currently, domestic research on the alleviating and / or treating effects of Lactobacillus curvaturei on vaginitis focuses on inhibiting pathogenic bacteria. There is a lack of evidence regarding its ability to improve inflammation through targeted host immune regulation, and there is also a lack of evidence regarding its effectiveness when taken orally. Summary of the Invention

[0006] This invention provides a strain of Lactobacillus curvaturei ( Lactobacillus crispatus CCFM1298, the *Lactobacillus curvatureii* ( Lactobacillus crispatus CCFM1298 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 9, 2022, with accession number GDMCC No: 62275.

[0007] The present invention provides *Lactobacillus curvatureii* ( Lactobacillus crispatus) CCFM1298 is derived from a healthy female vaginal swab in Jiangsu, the genome of CCFM1298 is extracted, the 16S rDNA of strain CCFM1298 is amplified and sequenced (by Suzhou Jinyuzhi Biological Technology Co., Ltd., the nucleotide sequence of 16S rDNA amplified from CCFM1298 is shown as SEQ ID NO. 1), the sequence is compared in NCBI nucleotide sequence, the result shows that the strain is lactobacillus crispatus, named lactobacillus crispatus (Lactobacillus crispatus) Lactobacillus crispatus ) CCFM1298.

[0008] The application also provides a microbial agent, wherein the microbial agent contains the above lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298 or a fermentation liquor thereof, or a freeze-dried powder containing the lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298.

[0009] In an embodiment of the application, the microbial agent contains the lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298, and the viable bacterial count of the lactobacillus crispatus (Lactobacillus crispatus 6 CFU / mL or 10 6 CFU / g.

[0010] In an embodiment of the application, the microbial agent is a powder obtained by drying a bacterial liquid containing the lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298, and the number of bacterial bodies is not less than 10 6 CFU / mL.

[0011] The application also provides a product containing the above lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298 or the above microbial agent.

[0012] In an embodiment of the application, the product contains the lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298, and the content of the lactobacillus crispatus (Lactobacillus crispatus 6 CFU / mL or 1×10 6 CFU / g.

[0013] In an embodiment of the application, the product is a medicine.

[0014] In an embodiment of the application, the medicine contains the lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298 and / or a fermentation product of the lactobacillus crispatus (Lactobacillus crispatus Lactobacillus crispatus ) CCFM1298 and a pharmaceutically acceptable carrier.

[0015] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0016] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0017] The present invention also provides the aforementioned *Lactobacillus curvatureii* ( Lactobacillus crispatus The use of CCFM1298 or the microbial agents described therein in the preparation of products for relieving and / or treating bacterial and candidal vaginitis.

[0018] In one embodiment of the present invention, the relief and / or treatment of bacterial vaginosis and / or candidal vaginitis includes modulating local host immunity, inhibiting the colonization of pathogenic bacteria, reducing the shedding of vaginal epithelial cells, and / or reducing the content of sialic acid sidase.

[0019] In one embodiment of the present invention, the product is a pharmaceutical product.

[0020] In one embodiment of the present invention, the pharmaceutical product comprises the *Lactobacillus curvatureii* (…). Lactobacillus crispatus CCFM1298 and / or the aforementioned Lactobacillus curvature ( Lactobacillus crispatus Fermentation products of CCFM1298 and pharmaceutically permissible carriers.

[0021] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0022] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0023] The present invention also provides the above-mentioned Lactobacillus curvatureus ( Lactobacillus crispatus The application of CCFM1298 or the above-mentioned microbial agents in the preparation of products that reduce vaginal epithelial shedding, alleviate vaginal tissue inflammation, and regulate local vaginal immunity.

[0024] Beneficial effects

[0025] (1) The present invention screened a strain of Lactobacillus curlis CCFM1298 from the vagina of healthy women. The Lactobacillus curlis alleviated the inflammatory response of BV and VVC by downregulating the expression of pro-inflammatory factors IL-1β and TNF-α and upregulating the expression of anti-inflammatory factor IL-10, thereby improving their symptoms and pathology. In addition, the Lactobacillus curlis CCFM1298 significantly reduced the shedding of vaginal epithelial cells.

[0026] (2) The Lactobacillus crispatus CCFM1298 provided by the application is derived from the vagina of a healthy female, and is applied to the preparation of a medicine for relieving vaginitis in females, so as to be more conducive to the recovery and balance of the vaginal microecosystem of females.

[0027] Biological material preservation

[0028] A Lactobacillus crispatus (CCFM1298) Lactobacillus crispatus , which is taxonomically named as: Lactobacillus crispatus , has been preserved in the Guangdong Microbial Culture Collection Center on December 9, 2022, with a preservation number of GDMCC No: 62275 and a preservation address of 5th Floor, Building 59, 100 Middle Martyrs Road, Guangzhou, Guangdong Microbial Institute. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a flow chart of the BV animal experiment design scheme.

[0030] Figure 2 It is a diagram of the regulation of CCFM1298 on the local immune system of the BV vagina; wherein (A) is the expression of the proinflammatory factor IL-1β; (B) is the expression of the proinflammatory factor TNF-α; compared with the model group, **: p <0.01, ***: p <0.001.

[0031] Figure 3 It is a diagram of the histopathological evaluation of CCFM1298 on the BV and VVC mouse vagina; wherein (A) is the blank control group; (B) is the bacterial vaginitis model group; (C) is the Lactobacillus crispatus CCFM1298 group.

[0032] Figure 4 It is the influence of CCFM1298 on the vaginal epithelial cell shedding of BV; compared with the model group, the blank control group and the Lactobacillus crispatus CCFM1298 group, ****: p <0.0001.

[0033] Figure 5 It is the influence of CCFM1298 on the concentration of sialic acid glycosidase in the vaginal secretion of BV; compared with the model group, the blank control group and the Lactobacillus crispatus CCFM1298 group, **: p <0.01, ***: p <0.001.

[0034] Figure 6 It is a flow chart of the VVC animal experiment design scheme.

[0035] Figure 7Figure 2 is a graph showing the regulation of the vaginal local immune system by CCFM1298 on VVC; wherein (A) expression of pro-inflammatory factor IL-1β; (B) expression of anti-inflammatory factor IL-10; compared with the model group, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. p <0.05, **: p <0.01, ***: p <0.001.

[0036] Figure 8 Figure 3 is a graph showing the histopathological evaluation of the vagina of a VVC mouse by CCFM1298; wherein (A): blank control group; (B): Candida vaginitis model group; (C): Lactobacillus crispatus CCFM1298 group.

[0037] Figure 9 Figures 4 and 5 are graphs showing the effect of CCFM1298 on the exfoliation of epithelial cells of VVC; **: p<0.01. p <0.01,, ****: p<0.0001. DETAILED DESCRIPTION

[0038] The Gardnerella vaginalis involved in the following examples is Gardnerella vaginalis ATCC 14018, which is purchased from Guangdong Microbial Institute Culture Collection Center (GDMCC). The Candida albicans involved in the following examples is isolated from the vaginal secretions of a patient with candida vaginitis and is preserved in the culture collection center of the Food Biotechnology Research Center of Jiangnan University.

[0039] The culture medium involved in the following examples is as follows:

[0040] MRS liquid medium ( / L): 10.0 g of proteose peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 2.0 g of anhydrous sodium acetate, 2.6 g of potassium phosphate dibasic heptahydrate, 2.0 g of diammonium hydrogen citrate, 0.25 g of manganese sulfate monohydrate, 0.5 g of magnesium sulfate heptahydrate, 1 mL of Tween 80 is added, 1000 mL of distilled water is added, the pH is adjusted to 6.2-6.4, and high-pressure sterilization is performed at 115°C for 20 min.

[0041] BHI liquid medium ( / L): 10.0 g of tryptone, 17.5 g of beef heart infusion powder, 5.0 g of sodium chloride, 2.5 g of sodium phosphate dibasic, 2.0 g of glucose, 1% additional yeast extract, 0.1% maltose, 0.1% glucose, the pH value is adjusted to 7.4. High-pressure sterilization is performed at 115°C for 20 min. Before use, 10% sterile fetal bovine serum is added to the culture medium.

[0042] YPD liquid medium ( / L): peptone 20.0 g, glucose 20.0 g, yeast extract 10.0 g. High pressure sterilization at 115 ℃ for 20 min.

[0043] The detection method involved in the following examples is as follows:

[0044] Determination of TNF-α and IL-1β concentrations:

[0045] At the end of the experiment, the vaginal tissue was stripped after the mice were sacrificed by cervical dislocation, and the vaginal tissue sample (20 mg) was placed in 200 μL of RIPA lysis buffer containing 2% (v / v) protease inhibitor cocktail and 2% (v / v) phosphatase inhibitor cocktail, and was broken by a high-throughput tissue grinder (65 Hz, 45 s / time, 6 times) to obtain a vaginal tissue homogenate. The vaginal tissue supernatant was centrifuged at 12000xg for 15 min at 4 ℃, and the determination of TNF-α and IL-1β concentrations was performed according to the kit instructions.

[0046] Detection of sialidase content

[0047] The vaginal lavage fluid of the mice was collected on day 18, and the cell debris was removed by centrifugation at 3600xg for 3 min at 4 ℃. 10 μL of the vaginal lavage fluid was extracted from the vaginal supernatant for the experiment. The sialidase content was determined by enzyme-linked immunosorbent assay (ELISA), and the OD value was compared with the standard curve. The specific operation was performed according to the kit instructions. 450 nm

[0048] Determination of IL-1β and IL-10 concentrations:

[0049] The vaginal tissue was stripped after the mice were sacrificed by cervical dislocation, and the vaginal tissue sample (20 mg) was placed in 200 μL of RIPA lysis buffer containing 2% (v / v) protease inhibitor cocktail and 2% (v / v) phosphatase inhibitor cocktail, and was broken by a high-throughput tissue grinder (65 Hz, 45 s / time, 6 times) to obtain a vaginal tissue homogenate. The vaginal tissue supernatant was centrifuged at 12000xg for 15 min at 4 ℃, and the determination of IL-1β and IL-10 concentrations was performed according to the kit instructions.

[0050] Example 1: Isolation and identification of Lactobacillus crispatus (L. crispatus) CCFM1298 Lactobacillus crispatus Isolation and identification method of L. crispatus CCFM1298

[0051] (1) Isolation

[0052] ​The sample was a healthy vaginal swab from a local woman in Jiangsu. After pretreatment, the sample was stored in 30% glycerol at -80°C in a refrigerator. After thawing, the sample was mixed and 0.5 mL of the sample was taken and diluted ten times in 4.5 mL of 0.9% physiological saline. The appropriate gradient diluent was selected and coated on MRS solid medium, which was incubated at 37°C for 48 h. The typical colonies of Lactobacillus crispatus were picked and streaked on MRS solid medium for purification. The single colonies were picked and inoculated in MRS liquid medium for enrichment. The bacterial cells were resuspended in 30% glycerol for preservation, and the strain CCFM1298 was obtained. The typical colonies of Lactobacillus crispatus were small, white and round, with a full center and a diffuse periphery.

[0053] (2) Identification

[0054] The genome of CCFM1298 was extracted, and the 16S rDNA of the strain CCFM1298 was amplified and sequenced (by Suzhou Jinyuzhi Biological Technology Co., Ltd., the nucleotide sequence of the 16S rDNA amplified from CCFM1298 is shown as SEQ ID NO. 1). The sequence was compared in NCBI, and the results showed that the strain was Lactobacillus crispatus, named Lactobacillus crispatus (Lactobacillus crispatus) CCFM1298. Lactobacillus crispatus

[0055] Example 2: Application of Lactobacillus crispatus CCFM1298 in alleviating bacterial vaginosis in mice

[0056] The specific implementation is as follows:

[0057] 1. Preparation of Lactobacillus crispatus CCFM1298 bacterial suspension and Gardnerella vaginalis bacterial suspension

[0058] (1) Lactobacillus crispatus CCFM1298 bacterial suspension:

[0059] After three generations of activation of Lactobacillus crispatus CCFM1298 preserved in 30% glycerol at -80°C in a refrigerator in MRS liquid medium, centrifugation was performed at 8000 g for 15 min, and the bacterial slurry of the activated three generations of Lactobacillus crispatus was collected. The bacterial slurry was washed twice with PBS and resuspended with PBS at 1:100. Finally, a Lactobacillus crispatus CCFM1298 bacterial suspension with a concentration of 1×10 10 CFU / mL was obtained for intervention experiments.

[0060] (2) Gardnerella vaginalis bacterial suspension:

[0061] ​The Gardnerella vaginalis preserved in a -80 °C refrigerator with 30% glycerol was activated for three generations in BHI liquid medium, centrifuged at 8000 g for 15 min, and the bacterial slurry of Gardnerella vaginalis after activation for three generations was collected. The bacterial slurry was washed twice with PBS and resuspended with PBS at 1:10. Finally, a Gardnerella vaginalis bacterial suspension with a concentration of 1 x 10 10 CFU / mL was obtained for the infection experiment.

[0062] 2. Animal experiment

[0063] (1) Experimental animals:

[0064] SPF level 7-week-old female BALB / c mice, weighing 17-19 g, were raised in an IVC system for one week. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Jing) 2012-0001). During the feeding period, each mouse was free to drink water and fed with SPF mouse feed (Co 60 sterilized). The feeding environment: half cycle lighting each day and night, temperature 22-24 °C, humidity about 45%-55%.

[0065] (2) The experimental process is shown in Figure 1 .

[0066] Experimental scheme and grouping: the mice were randomly divided into 3 groups according to body weight, as shown in Table 1.

[0067] Table 1: Mouse experimental grouping

[0068]

[0069] The experimental period was 22 days (days -3-18), and before the experiment, the mice were in an adaptation period for one week. Three days before infection and on the day of infection, all mice except the blank group were subcutaneously injected with 100 μL of estradiol valerate (0.5 mg of estradiol valerate dissolved in 100 μL of sesame oil) to induce estrus, which was performed every morning, once a day for 7 days, and continued until the end of the experiment.

[0070] Infection experiment (modeling period):

[0071] On day 0, the mice in the blank control group were inoculated with 20 μL of PBS buffer in the vagina, and the rest of the mice were inoculated with 20 μL of Gardnerella vaginalis bacterial suspension with a concentration of 10 10 CFU / mL in the vagina. After inoculation, the mice were inverted for 1-2 minutes to prevent bacteria from flowing out, and the Gardnerella vaginalis inoculation was continued for 7 days, until the end of the 6th day.

[0072] Intervention experiment:

[0073] From the 7th day, the blank control group mice: 200 μL of physiological saline was given by gavage once a day;

[0074] The model group mice: 200 μL of physiological saline was given by gavage once a day;

[0075] The CCFM1298 group mice: 200 μL of CCFM1298 bacterial suspension with a concentration of 10 9 CFU was given by gavage once a day;

[0076] The intervention of the above groups was once a day, for 12 consecutive days.

[0077] At the end of the infection (7th day) and the end of the intervention (18th day), the mice were sampled by blowing the vaginal head with 50 μL of phosphate buffer solution each time, and finally 300 μL of vaginal lavage fluid was collected to determine the Gardnerella vaginalis load, sialidase content and vaginal epithelial cell shedding number. At the same time, on the 19th day, all the experimental mice were sacrificed and the vaginal tissue was stripped for subsequent histopathological analysis to determine the secretion of inflammatory factors (TNF-α, IL-1β).

[0078] 3. Experimental results:

[0079] (1) Effect of CCFM1298 on the local immunity of bacterial vaginosis mice

[0080] At the end of the experiment, the mice were sacrificed by cervical dislocation and the vaginal tissue was stripped. The supernatant of the vaginal tissue was determined for TNF-α and IL-1β concentration according to the instructions of the kit, and the results are shown in Figure 2 Figure 2 A and 2B show the concentrations of pro-inflammatory factors IL-1β and TNF-α, respectively.

[0081] The results show that:

[0082] The blank group: the concentrations of pro-inflammatory factors IL-1β and TNF-α were 121.7 pg / mg and 115.8 pg / mg, respectively.

[0083] The model group: after the vaginal infection of Gardnerella vaginalis, the host's natural immune system was stimulated, and the concentrations of pro-inflammatory factors IL-1β and TNF-α increased by about 3 times compared with the blank control (316 pg / mg, p <0.001; 378.6 pg / mg, p <0.01).

[0084] The CCFM1298 group: after 12 days of CCFM1298 intervention, the concentrations of pro-inflammatory factors IL-1β and TNF-α were significantly reduced (120.7 pg / mg, p <0.001; 177.9 pg / mg,​p <0.01), and the inhibition rates were 61.8% and 53%, respectively.

[0085] (2) Histopathological analysis of vaginal tissue of mice

[0086] The vaginal tissue of mice was fixed and preserved with 4% paraformaldehyde solution, and then paraffin-embedded, 5 mm-thick sectioned, and stained with hematoxylin and eosin (H&E). The section specimen was observed under a pathological section scanner at 30 times magnification, and the results are shown in Figure 3 Figure 3 A, 3B and 3C show the HE staining images of the blank control group, the bacterial vaginitis model group and the CCFM1298 intervention group, respectively.

[0087] The results show that:

[0088] Blank group: The surface of the vaginal tissue stratum spinosum was smooth and continuous, no necrosis and shedding, no obvious inflammatory cell infiltration was found, and the connection between the stratum spinosum cells was tight, and no obvious intercellular bridge structure was found.

[0089] Model group: Compared with the blank control, the surface of the vaginal tissue stratum spinosum was severely damaged, and obvious necrosis and shedding occurred, and a large number of neutrophilic granulocytes infiltrated to form microabscesses. The stratum spinosum cells were loose, and obvious intercellular bridge structure was observed, and a large number of inflammatory cells infiltrated the dermis.

[0090] CCFM1298 group: After 12 days of CCFM1298 intervention, compared with the model group, the inflammatory cell infiltration phenomenon on the surface of the stratum spinosum was obviously improved, the continuity was obviously improved, and the intercellular structure of the stratum spinosum cells was gradually tight.

[0091] (3) Effect of CCFM1298 on vaginal epithelial cell shedding caused by Gardnerella vaginalis

[0092] 10 μL sample was transferred from the vaginal lavage fluid of the 18th day mouse to a glass slide, and 10 μL gun head outer wall was gently smeared. After the specimen was fixed with a fixative, DiffQuik staining was performed according to the kit instructions. Under a 400 times optical microscope, the number of epithelial cell shedding in 5 microscopic fields was randomly counted, and the results are shown in Figure 4 .

[0093] The results show that Gardnerella vaginalis infection causes a large number of vaginal epithelial cell shedding (36.62±2.53 / field), and the blank control group has only 6.72±2.1 epithelial cells shedding per field p <0.0001).

[0094] After 12 days of CCFM1298 intervention, the number of vaginal epithelial cell shedding can be significantly reduced (10.64±0.93 / field) p ​<0.0001).

[0095] (4) Effect of CCFM1298 on sialate content in mice with bacterial vaginosis

[0096] On day 18, the mouse vaginal lavage fluid was collected, and the sialate content was determined by enzyme-linked immunosorbent assay (ELISA). The OD value was compared with the standard curve, and the results are shown in 450 nm Figure 5

[0097] The results showed that the blank group: the concentration of sialate in the mouse vaginal secretion was 43.3 ng / L.

[0098] The model group: after the mouse vaginal infection with Gardnerella vaginalis, the sialate in the mouse vaginal secretion was significantly increased to 59.7 ng / L (P < 0.001). p <0.001).

[0099] The CCFM1298 group: after 12 days of intervention of CCFM1298, the concentration of sialate was significantly reduced to 50.7 ng / L (P < 0.01), indicating that CCFM1298 can significantly reduce the concentration of sialate. p <0.01).

[0100] Example 3: Application of Lactobacillus crispatus CCFM1298 in relieving mouse candidal vaginitis

[0101] The specific implementation is as follows:

[0102] 1. Preparation of Lactobacillus crispatus CCFM1298 bacterial suspension and Candida albicans bacterial suspension

[0103] (1) Preparation of Lactobacillus crispatus CCFM1298 bacterial suspension:

[0104] After three generations of activation of Lactobacillus crispatus CCFM1298 preserved in a -80°C refrigerator with 30% glycerol in MRS liquid medium, centrifugation at 8000 g for 15 min, the bacterial slurry of the activated three generations of Lactobacillus crispatus bacterial suspension was collected. The bacterial slurry was washed twice with PBS and resuspended with PBS at 1:100. Finally, the concentration of Lactobacillus crispatus CCFM1298 bacterial suspension was 1×10 10 CFU / mL, which was used for intervention experiments.

[0105] (2) Candida albicans bacterial suspension

[0106] ​​C. albicans preserved in -80°C refrigerator with 30% glycerol was activated for three generations in YPD liquid medium, and then centrifuged at 8000 g for 15 min to collect the activated C. albicans slurry. The slurry was washed twice with PBS and resuspended with PBS at 1:10. Finally, a C. albicans bacterial suspension with a concentration of 1×10 8 CFU / mL was obtained for the infection experiment.

[0107] 2. Animal experiment

[0108] (1) Experimental animals:

[0109] SPF level 7-week-old female BALB / c mice, weighing 17-19 g, were raised in an IVC system for one week of adaptation. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Jing) 2012-0001). During the feeding period, each mouse had free access to water and was fed with SPF mouse feed (Co 60 sterilized). The feeding environment: half cycle lighting each day and night, temperature 22-24°C, humidity about 45%-55%.

[0110] (2) The experimental process is shown in Figure 6 .

[0111] Experimental scheme and grouping: the mice were randomly divided into 3 groups according to body weight, as shown in Table 2.

[0112] Table 2 Mice experimental grouping

[0113]

[0114] The experimental period was 22 days (days -3-18), and before the experiment, the mice were in a one-week adaptation period. Three days before infection and on the day of infection, all mice except the blank group were subcutaneously injected with 100 μL of estradiol valerate (0.5 mg of estradiol valerate dissolved in 100 μL of sesame oil) to induce estrus, which was performed every morning, once a day for 7 days, and continued until the end of the experiment.

[0115] Infection experiment (modeling period):

[0116] On day 0, the mice in the blank control group were inoculated with 20 μL of PBS buffer in the vagina, and the rest of the mice were inoculated with 20 μL of C. albicans bacterial suspension with a concentration of 10 8 CFU / mL in the vagina. After inoculation, the mice were inverted for 1-2 minutes to prevent the bacterial solution from flowing out, and the C. albicans inoculation was continued for 7 consecutive days until the end of the 6th day.

[0117] Intervention experiment:

[0118] From the 7th day, the blank control group mice: once a day, 200 μL of normal saline was given by gavage;

[0119] The model group mice: once a day, 200 μL of normal saline was given by gavage;

[0120] The CCFM1298 group mice: once a day, 200 μL of CCFM1298 with a concentration of 10 9 CFU of Lactobacillus CCFM1298 was given by gavage;

[0121] The intervention of each group was once a day, for 12 consecutive days.

[0122] At the end of the infection (7th day) and the end of the intervention (18th day), the mice were sampled by blowing 50 μL of phosphate buffer solution into the vagina with a gun head each time, and finally 350 μL of vaginal lavage fluid was collected to determine the Candida albicans load and the number of vaginal epithelial cell exfoliation. At the same time, on the 19th day, all the experimental mice were sacrificed and the vaginal tissue was stripped for subsequent histopathological analysis to determine the secretion of inflammatory factors (IL-1β, IL-10).

[0123] 3. Experimental results:

[0124] (1) Effect of CCFM1298 on the local immunity of mice with Candida vaginitis

[0125] At the end of the experiment, the mice were sacrificed by cervical dislocation and the vaginal tissue was stripped, and the vaginal tissue supernatant was determined for IL-1β and IL-10 concentration according to the kit instructions, the results are shown in Figure 7 Figure 7 A and Figure 7 B show the concentration of pro-inflammatory factor IL-1β and anti-inflammatory factor IL-10, respectively.

[0126] The results show that:

[0127] Model group and blank group: when Candida albicans infects the vagina (model group), the expression level of pro-inflammatory factor IL-1β in the model group (323.6 pg / mg) is 2.3 times higher than that in the blank control group (141.5 pg / mg) (P p <0.001); the expression level of anti-inflammatory factor IL-10 in the model group (268 pg / mg) is 34.3% lower than that in the blank control group (408 pg / mg).

[0128] After CCFM1298 intervention, the expression level of IL-1β was significantly reduced to 229.8 pg / mg (P p <0.05), with an inhibition rate of 28.9%; the expression level of anti-inflammatory factor IL-10 was significantly increased to 491.3 pg / mg (P p ​<0.05), increased by 83.3%; it was shown that CCFM1298 could effectively regulate the expression of inflammatory factors in mice, and reduce inflammation caused by Candida albicans.

[0129] (2) Histopathological analysis of vaginal tissue of mice

[0130] The vaginal tissue of mice was fixed and preserved with 4% paraformaldehyde solution, and after paraffin embedding, 5 mm thick sections were cut and stained with hematoxylin and eosin (H&E). The section specimen was observed under a pathological section scanner at 30 times magnification, and the results are shown in Figure 8 Figure 8 A, 8B and 8C show the HE staining images of the blank control group, the Candida vaginitis model group and the CCFM1298 group, respectively.

[0131] The results show that:

[0132] The blank group: the surface of the vaginal tissue stratum spinosum was smooth and continuous, without necrosis and shedding, and no obvious inflammatory cell infiltration was found, and the stratum spinosum cells were tightly connected, and no obvious intercellular bridge structure was found.

[0133] The model group: compared with the blank control, the vaginal tissue stratum spinosum was obviously edematous, the keratinocytes were degenerated and destroyed into a reticular shape, a large number of eosinophils, neutrophils and lymphocytes were distributed therein, and the stratum spinosum was loose, and the number of intercellular bridges increased.

[0134] The CCFM1298 group: after 12 days of CCFM1298 intervention, compared with the model group, the inflammatory cell infiltration was obviously improved, the epithelium gradually became smooth, the stratum spinosum cell connection gradually became tight, and only part of the mucosa was damaged.

[0135] (3) Effect of CCFM1298 on vaginal epithelial cell shedding caused by Candida albicans

[0136] 10 μL samples were transferred to a glass slide from the 18th day of the mouse vaginal lavage fluid, and 10 μL of the outer wall of the gun head was gently rubbed. After the specimen was fixed with a fixative, DiffQuik staining was performed according to the kit instructions. Under a 400x optical microscope, the number of epithelial cell shedding in 5 microscopic fields was randomly counted, and the results are shown in Figure 9 .

[0137] The results show that Candida albicans infection causes a large number of vaginal epithelial cell shedding (36.2±5.49 / field), and the blank control group has only 8.4±2.7 epithelial cells shedding per field (P<0.0001), and CCFM1298 can significantly reduce the number of vaginal epithelial cell shedding (23.94±3.53 / field) after 12 days of intervention (P<0.01). p< p< ​​​

[0138] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is to be understood that the application is not limited to those precise embodiments, and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope or spirit of the application. Therefore, the scope of the present application should be limited only by the appended claims.

Claims

1. A Lactobacillus crispatus (L. crispatus) strain CCFM1298, deposited with the Guangdong Microbial Culture Collection Center on December 9, 2022, and assigned accession number GDMCC No: 62275. Lactobacillus crispatus ) CCFM1298, deposited with the Guangdong Microbial Culture Collection Center on December 9, 2022, and assigned accession number GDMCC No: 62275.

2. A microbial inoculant, characterized in that, The microbial agent contains the Lactobacillus crispatus CCFM1298 or the fermentation broth thereof according to claim 1, or contains the freeze-dried powder of the Lactobacillus crispatus CCFM1298.

3. The microbial inoculant of claim 2, wherein, The viable cell number of Lactobacillus crispatus CCFM1298 in the microbial inoculant is not less than 10 6 CFU / mL or 10 6 CFU / g.

4. A product characterized by, The product contains the Lactobacillus crispatus CCFM1298 according to claim 1 or the microbial agent according to claim 2 or 3, and the product is a pharmaceutical product.

5. The product of claim 4, wherein, The pharmaceutical product contains the Lactobacillus crispatus CCFM1298 according to claim 1 and / or the fermentation broth of the Lactobacillus crispatus CCFM1298 and a pharmaceutically acceptable carrier.

6. Use of Lactobacillus crispatus CCFM1298 according to claim 1 or of the microbial agent according to claim 2 or 3 for the manufacture of a product for the relief and / or treatment of bacterial and candidal vaginitis, characterized in that, The product is a pharmaceutical product, and the bacteria are Gardnerella vaginalis.

7. Use according to claim 6, characterized in that, The pharmaceutical product contains the Lactobacillus crispatus CCFM1298 according to claim 1 and / or the fermentation broth of the Lactobacillus crispatus CCFM1298 and a pharmaceutically acceptable carrier.

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