Lactobacillus johnsonii for improving female intimate health and skin aging simultaneously

Abstract

The application discloses lactobacillus johnsonii capable of orally assisting in relieving vaginitis and improving skin aging, and belongs to the technical field of microorganisms.The lactobacillus johnsonii is separated from vaginal secretions of healthy women, has the effects of repairing immune disorders caused by vaginitis and skin aging of mice with vaginitis, and is specifically reflected in the following aspects: reducing the content of IL-1beta in cell supernatant; improving inflammatory reactions of mouse vaginas; reducing the expression of TLR-4 mRNA and the level of TNF-alpha in vaginal tissues; enhancing the anti-aging ability of mouse skin; reducing the expression of MAPK / AP-1 / MMP-1 mRNA, increasing the content of Col I and Col III in skin; improving pathological characteristics of mouse vaginas and reducing the load of pathogenic bacteria.Therefore, the lactobacillus johnsonii has great application prospects in products for improving and repairing inflammatory damage caused by vaginitis and skin health problems of mice with vaginitis.

Description

TECHNICAL FIELD

[0001] The present application relates to a Lactobacillus jensenii capable of alleviating vaginitis through oral administration while improving skin aging, belonging to the field of microbial technology. BACKGROUND

[0002] The dominant microorganism in the vagina of healthy women is mainly lactobacillus. When the vaginal environment changes, causing lactobacillus to be replaced by facultative anaerobes or anaerobes such as Gardnerella vaginalis, it leads to vaginosis, a common vaginal infectious disease among women of childbearing age.

[0003] The vaginal epithelial cells and immune cells wandering in the vaginal tissue first contact the pathogenic microorganisms such as Gardnerella vaginalis, and the microbial pathogen molecules bind to Toll-like receptors (TLRs), secreting pro-inflammatory cytokines such as TNF-α and IL-1β, causing the immune response of the vaginal mucosa to Gardnerella vaginalis and accompanying tissue inflammation. Local immune response can affect the overall health of the whole body and other parts of the body through the circulatory system, and activated immune cells wander to the skin tissue, secrete pro-inflammatory factors, cause abnormal expression of collagen-degrading enzymes, promote the decrease of collagen content, and lead to skin aging.

[0004] Currently, the main clinical treatment for vaginitis includes the use of antibacterial drugs such as nitroimidazoles (metronidazole, tinidazole) and lincomycin (clindamycin). However, antibacterial drugs cannot regulate the immune response of the vagina, which can easily lead to the recurrence of vaginitis. At the same time, studies have shown that the use of Lactobacillus live capsules in the treatment of recurrent vaginitis can significantly reduce the recurrence rate. The main component of the prescription drug "Dingjunsheng" for treating vaginitis is Lactobacillus delbrueckii DM8909, which has been shown to have good probiotic properties, such as certain adhesion advantages to vaginal epithelial cells, and can inhibit the growth and reproduction of harmful pathogenic bacteria, regulate the balance of vaginal flora, and thus effectively treat bacterial vaginosis (Study on the inhibition of Lactobacillus DM8909 strain on vaginal infection in mice, Chinese Journal of Microecology, 2003).

[0005] In addition, the patents with publication numbers CN110777087A and CN110656060A have disclosed the inhibitory ability of Lactobacillus jensenii on vaginal disease-causing bacteria, and its good cell adhesion ability. However, it is unknown whether the above-mentioned strains can stimulate the immune response, and it is also unclear how they affect the skin health of mice with vaginitis. Therefore, there is currently a lack of Lactobacillus jensenii that can alleviate vaginitis by regulating the immune disorder caused by vaginitis, and can also improve skin health at the same time. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a Lactobacillus johnsonii strain that can improve female private health and skin aging at the same time, aiming to solve the technical problem that there is a lack of Lactobacillus johnsonii that can relieve vaginitis by regulating immune disorders caused by vaginitis and can also improve skin health in the prior art.

[0007] The first technical solution provided by the present application is a Lactobacillus johnsonii CCFM1314, which was deposited in the Guangdong Microbial Culture Collection Center on August 4, 2023, and the deposit number is GDMCCNo:63710. The Lactobacillus johnsonii CCFM1314 is isolated from the vaginal secretions of healthy women.

[0008] The second technical solution provided by the present application is a microbial preparation containing the Lactobacillus johnsonii CCFM1314 of the first technical solution.

[0009] In some embodiments, the content of the Lactobacillus johnsonii CCFM1314 in the microbial preparation is not less than 1x10 6 CFU / mL or 1x10 6 CFU / g.

[0010] Further, the content of the Lactobacillus johnsonii CCFM1314 in the microbial preparation is not less than 1x10 9 CFU / mL or 1x10 9 CFU / g.

[0011] The third technical solution provided by the present application is a product containing the Lactobacillus johnsonii CCFM1314 of the first technical solution or the microbial preparation of the second technical solution.

[0012] In some embodiments, the product is a pharmaceutical product or a health product.

[0013] Further, the pharmaceutical product comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0014] Further, the carrier includes one or more of fillers, binders, humectants, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0015] Further, the dosage form of the pharmaceutical product includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0016] Further, the pharmaceutical product includes enteric-coated tablets and capsules, oral liquids, vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0017] Further, the health products include health wet paper, sanitary napkin, sanitary pad, sanitary plug, sanitary cotton, vaginal wash, women's antibacterial / antibacterial lotion.

[0018] In some embodiments, the Lactobacillus johnsonii CCFM1314 is present in the product in an amount of not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0019] Further, the Lactobacillus johnsonii CCFM1314 is present in the product in an amount of not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0020] The four technical solutions provided by the present application are the use of the Lactobacillus johnsonii CCFM1314 of the first technical solution or the microbial preparation of the second technical solution in the preparation of products for relieving and / or treating vaginitis.

[0021] In some embodiments, the effect of relieving and / or treating vaginitis includes reducing the inflammatory response of vaginal tissue by reducing the expression of TLR-4 mRNA in vaginal tissue and the level of TNF-α in vaginal tissue, and repairing the immune disorder of vaginal tissue caused by vaginitis.

[0022] In some embodiments, the product is a pharmaceutical product or a health product.

[0023] Further, the pharmaceutical product comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0024] Further, the carrier includes one or more of the fillers, binders, humectants, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0025] Further, the dosage form of the pharmaceutical product includes granules, capsules, tablets, pills, suppositories, or oral liquids.

[0026] Further, the pharmaceutical product includes enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, gels.

[0027] Further, the health product includes health wet paper, sanitary napkin, sanitary pad, sanitary plug, sanitary cotton, vaginal wash, women's antibacterial / antibacterial lotion.

[0028] The five technical solutions provided by the present application are the use of the Lactobacillus johnsonii CCFM1314 of the first technical solution or the microbial preparation of the second technical solution in the preparation of products for relieving and / or treating skin aging caused by vaginitis.

[0029] In some embodiments, the effect of alleviating and / or treating vaginitis-induced skin aging comprises down-regulating MAPK / AP-1 / MMP-1 mRNA expression in skin tissue, and increasing Col I and Col III contents in skin tissue.

[0030] MAPK is an intracellular threonine protein kinase, which can be activated by reactive oxygen species (ROS) released from damaged vaginal epithelial cells, thereby further regulating the transcription of MMPs, especially interstitial collagenase (MMP-1), stromelysin-1 (MMP-3) and gelatinase (MMP-9) of the MMP family, leading to the degradation of collagen and elastin. Among them, the collagen in human skin is mainly type I and type III collagen, in which type I collagen (Col I) accounts for about 70% to 80% of the dry weight of skin collagen, and is an important basis for skin to withstand tension and maintain skin fullness; type III collagen (Col III) accounts for about 10%, and is the main component of reticular fibers.

[0031] In some embodiments, the product is a pharmaceutical product or a hygiene product.

[0032] Further, the pharmaceutical product comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0033] Still further, the carrier comprises one or more of a filler, a binder, a humectant, a disintegrant, a lubricant, a flavoring agent commonly used in medicine.

[0034] Further, the dosage form of the pharmaceutical product comprises granules, capsules, tablets, pills, suppositories or oral liquids.

[0035] Still further, the pharmaceutical product comprises enteric-coated tablets and capsules for oral administration, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, gels.

[0036] Further, the hygiene product comprises a sanitary wet tissue, a sanitary napkin, a sanitary pad, a sanitary plug, a sanitary cotton, a vaginal wash, a lady's antibacterial / antifungal lotion.

[0037] Compared with existing technologies, the present invention has the following technical effects: The present invention provides a strain of Lactobacillus johnsonii CCFM1314, isolated from the vaginal secretions of healthy women, which has the effect of repairing immune disorders caused by vaginitis and skin aging in mice with vaginitis, specifically manifested in: (1) reducing the content of IL-1β in cell supernatant; (2) improving the vaginal inflammatory response in mice: reducing TLR-4 mRNA expression and vaginal tissue TNF-α level; (3) enhancing the anti-aging ability of mouse skin: downregulating MAPK / AP-1 / MMP-1 mRNA expression and increasing the content of skin ColⅠ and ColⅢ; (4) improving the pathological features of mouse vagina and reducing the pathogenic bacterial load. Therefore, Lactobacillus johnsonii CCFM1314 has great application prospects in products that improve and repair inflammatory damage caused by vaginitis and skin health problems in mice with vaginitis.

[0038] Preservation of biological materials

[0039] The Lactobacillus johnsonii CCFM1314 provided by this invention was isolated from vaginal secretions of a healthy woman and deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo:63710, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0040] Figure 1 Effect of different bacterial lysates on the concentration of VK2 / E6E7 IL-1β in vaginal epithelial cells; compared with the model group, *p<0.05, **p<0.01.

[0041] Figure 2 Animal experiment design flowchart.

[0042] Figure 3 Figure: Effect of Lactobacillus johnsonii on vaginal immune response; (a) Effect of vaginal tumor necrosis factor TNF-α concentration; (b) Effect of TLR-4 mRNA expression, compared with the model group, ****p<0.0001.

[0043] Figure 4 Effects of Lactobacillus johnsonii on skin aging: (a) Effect on MAPK mRNA expression; (b) Effect on AP-1 mRNA expression; (c) Effect on MMP-1 mRNA expression. Compared with the model group, **p<0.01, ***p<0.001, ****p<0.0001.

[0044] Figure 5Figure of the effect of L. johnsonii on the collagen content; (a) expression of Col I concentration; (b) Col III concentration. Compared with the model group, **p<0.01, ****p<0.0001.

[0045] Figure 6 Figure of the colonization of G. vaginalis in the vagina; G. vaginalis load in the vagina of BV mice on day 7 and day 17; a, b represent significant differences between different groups on day 17; p<0.01.

[0046] Figure 7 Figure of the histopathological evaluation of the vagina of mice. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below with reference to specific examples and with reference to the accompanying drawings.

[0048] The culture media involved in the following examples are as follows:

[0049] MRS culture 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, citric acid hydrogen diamine 2.0 g / L, potassium phosphate dibasic 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.

[0050] BHI culture medium: tryptone 10.0 g / L, beef heart infusion powder 17.5 g / L, sodium chloride 5.0 g / L, glucose 3.0 g / L, dodecahydrate sodium phosphate dibasic 2.5 g / L, yeast powder 10.0 g / L, maltose 1.0 g / L, pH 7.2-7.4; when the temperature cools to about 55°C, add 10% sterile fetal bovine serum.

[0051] 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 10000 U / mL, streptomycin concentration 10 mg / mL in the mixed solution).

[0052] The strains, cells and animals involved in the following examples are as follows:

[0053] SPF BALB / c mice, female, 7 weeks old, body weight 18-20 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Beijing) 2012-0001).

[0054] Gardnerella vaginalis ATCC 14018 was purchased from Guangdong Microbial Culture Collection Center (GDMCC).

[0055] Human vaginal epithelial cells VK2 / E6E7 were kindly gifted by People's Hospital, Obstetrics and Gynecology Department.

[0056] Lactobacillus gasseri QJSWX195M1 was preserved in Jiangnan University Food Microorganism Culture Collection Center.

[0057] The bacterial liquid involved in the following examples is shown as follows:

[0058] Lactobacillus johnsonii CCFM1314 liquid: Lactobacillus johnsonii CCFM1314 was inoculated in MRS liquid medium at 2% inoculation amount, and after 24h culture in a 37℃ incubator, the bacterial suspension concentration was adjusted to ~10 9 CFU / mL.

[0059] Gardnerella vaginalis suspension: Gardnerella vaginalis strain ATCC 14018 was inoculated in BHI medium, and cultured in a 37℃ incubator for 24h, and the bacterial suspension concentration was adjusted to 10 10 CFU / mL.

[0060] Example 1 Isolation and identification of Lactobacillus johnsonii CCFM1314

[0061] The specific steps are as follows:

[0062] 1. Screening

[0063] The sample was derived from vaginal secretion of healthy adult women, and after pretreatment, the sample was stored in 30% glycerol in a-80℃ refrigerator. After thawing, the sample was mixed and 0.5mL of the sample was taken and added to 4.5mL of physiological saline, and gradient dilution was performed with 9g / L physiological saline. The appropriate gradient dilution liquid was coated on MRS solid culture medium, and cultured at 37℃ for 48h. Typical colonies of Lactobacillus johnsonii were picked and streaked on MRS solid culture medium for purification, and single colonies were transferred to MRS liquid medium for enrichment, and stored in 30% glycerol, to obtain Lactobacillus johnsonii CCFM1314; wherein the typical colonies of Lactobacillus johnsonii were round and milky white.

[0064] 2. Identification

[0065] The genome of the strain CCFM1314 was extracted, the 16S rDNA of the strain was amplified and sequenced (the nucleotide sequence of the 16S rDNA amplified from CCFM1314 by Shanghai Meiji Biomedicine Technology Co., Ltd. is shown as SEQ ID NO. 1), and then the nucleotide sequence of the 16S rDNA amplified from the strain was subjected to nucleic acid sequence alignment in NCBI, and the results showed that the strain was Lactobacillus johnsonii, which was named Lactobacillus johnsonii CCFM1314.

[0066] Example 2: Lactobacillus down-regulates the level of IL-1β of vaginal epithelial cells

[0067] MRS solid culture medium was used to culture single colonies in a water-sealed constant temperature incubator at 37°C for 24-48h; the single colonies were picked and inoculated into MRS liquid culture medium, and cultured at 37°C for 12-18h to obtain culture solution 1; the culture solution 1 was inoculated into MRS liquid culture medium at an inoculation amount of 2% (v / v), and cultured at 37°C for 12h to obtain seed solution; the seed solution was inoculated into MRS liquid culture medium at an inoculation amount of 3-5% (v / v) for expansion culture, and cultured at a temperature of 37°C for 18-24h to obtain bacterial solution with a concentration of 3.4×10 8 CFU / mL. The bacterial solution was subjected to high-pressure homogenization (800-1200MPa, 5 times) to obtain bacterial lysate.

[0068] VK2 / E6E7 cells were inoculated into a 12-well plate at a concentration of 5×10 4 After the cells were cultured for 24h, a control group and a treatment group were set up; the treatment group was added with serum-free medium containing LPS at a concentration of 1.25μg / mL, and the control group was added with complete medium; after 24h, the old medium was discarded, and the cells were washed with PBS for 3 times; the control group was added with 5% MRS medium; the treatment group was added with bacterial lysate in the same proportion; 2mL of culture medium containing bacterial lysate of Lactobacillus johnsonii CCFM1314, Lactobacillus delbrueckii DM8909 or Lactobacillus gasseri QJSWX195M1 was respectively taken and added into the 12-well plate, and the cells were cultured for 24h, with three parallel samples for each sample. After the co-culture was completed, the culture supernatant was collected, and the content of IL-1β in the culture supernatant of the vaginal epithelial cells was detected by ELISA.

[0069] Figure 1The results show that compared with the control group IL-1β content 26.44 ng / L, the model group is 34.62 ng / L, the IL-1β content is significantly decreased, the IL-1β content after the intervention of Lactobacillus delbrueckii DM8909, Lactobacillus johnsonii CCFM1314 and Lactobacillus gasseri QJSWX195M1 is 30.23 ng / L, 29.57 ng / L and 33.23 ng / L respectively, compared with other groups (Lactobacillus delbrueckii DM8909 and Lactobacillus gasseri QJSWX195M1), the IL-1β content of the group intervened by Lactobacillus johnsonii CCFM1314 is significantly decreased (p<0.05).

[0070] Example 3: Application of Lactobacillus johnsonii CCFM1314 in relieving mouse vaginitis and skin aging

[0071] Figure 2 The experimental procedure is as follows:

[0072] Table 1 Experimental scheme and grouping:

[0073] Grouping Induced estrus Induced Gardnerella infection time Probiotic intervention time Blank control - - - Vaginitis model Day 1-3 Day 3-7 Day 8-17 Lactobacillus delbrueckii DM8909 Day 1-3 Day 3-7 Day 8-17 Lactobacillus johnsonii CCFM1314 Day 1-3 Day 3-7 Day 8-17 Lactobacillus gasseri QJSWX195M1 Day 1-3 Day 3-7 Day 8-17

[0074] According to the weight, the mice are randomly divided into 5 groups, referring to Table 1, all groups of mice are normally fed during the whole experiment.

[0075] Animal model construction: SPF level BALB / c mice, female, 7 weeks old, weight 18-20g are selected. After the adaptation period, the mice are injected with valeric acid estradiol for three consecutive days for induction of estrus treatment. The mice are continuously infected with Gardnerella vaginalis for 5 days once a day. The specific operation method of the infection is as follows: 10 10 CFU / mL of Gardnerella vaginalis bacterial suspension, the dose is 20μL per mouse. 20μL of bacterial suspension is sucked with a gun head and slowly injected into the mouse vagina. The mouse is inverted and stays for 1-2 minutes and put into the cage.

[0076] The mice with vaginitis model are divided into model (model) group and intervention group, wherein the intervention group is gavaged with 100μL of Lactobacillus johnsonii CCFM1314 bacterial suspension with a concentration of 10 9CFU / mL of probiotic bacteria, the model group was simply infected with G. vaginalis and did not receive subsequent intervention, and the intervention group was infected with G. vaginalis and then received subsequent intervention with probiotic bacteria. In addition, a control group was set up, in which the control group was not infected with G. vaginalis and did not receive subsequent intervention with probiotic bacteria. The vaginitis model group and the intervention group both experienced 3 consecutive days (days 1-3) of estrus induction by subcutaneous injection of 100 μL of estradiol valerate solution (0.5 mg of estradiol valerate dissolved in 100 μL of filtered and sterilized sesame oil) in the neck. The mice were infected with G. vaginalis for 5 consecutive days (days 3-7) (the specific method of infection was to use a gun to suck 20 μL of a suspension of G. vaginalis and slowly inject it into the mouse vagina, with the mouse being held upside down for 1-2 minutes and then placed in a cage). 9 CFU / mL of Lactobacillus (days 8-17). At the end of the infection (day 7) and the end of the intervention (day 17), 50 μL of phosphate buffer solution was used to sample the mouse vagina by blowing and sucking with a gun, and a total of 300 μL of vaginal lavage fluid was collected to determine the load of G. vaginalis and Lactobacillus. At the same time, on day 18, all the experimental mice were sacrificed and the vaginal tissue and dorsal skin tissue were removed for subsequent histopathological analysis to detect the colonization of G. vaginalis in the mouse vagina, the shedding of vaginal epithelial cells, the secretion of tumor necrosis factor (TNF-α), the expression of inflammatory receptors (TLR-4), the expression of MAPK / AP-1 / MMP-1 mRNA in the skin tissue of the mice, and the content of Col I and Col III.

[0077] Results of the experiment:

[0078] (1) TNF-α secretion and TLR-4 mRNA expression in mouse vaginal tissue

[0079] Twenty mg of vaginal tissue was taken from each of the five groups of mice to determine the cytokine, and 180 μL of pre-cooled PBS was used to homogenize the vaginal tissue. The sample was centrifuged at 3000 r / min for 15 min at 4°C, and the vaginal tissue supernatant was used to determine the concentration of TNF-α according to the instructions of the kit (Nanjing Senbeijia Biological Technology Co., Ltd.). The total RNA of the mouse vaginal tissue was extracted by the Trizol method, and the OD 260 / OD 280The total RNA was reverse transcribed into 20 μL cDNA by reverse transcription kit, and the fluorescence quantitative PCR reaction system was configured by ChamQ Universal SYBR qPCR Master Mix. GAPDH was used as the internal reference gene, and the expression of target genes was calculated based on 2 -ΔΔCt The expression of target genes was calculated by the method, and the primers are shown in Table 2.

[0080] Table 2 Primers and sequences

[0081]

[0082]

[0083] The results are shown in Figure 3 (a) shows the concentration of TNF-a in the vaginal tissue. The results show that the secretion of TNF-a in the vaginal tissue of the model group mice is greatly increased compared with the control group, and the concentration is 239.97 ng / L. After the intervention of probiotics, the inflammatory factors of all groups are significantly down-regulated, and compared with Lactobacillus delbrueckii DM8909 (down-regulated by 13.98% compared with the model group), Lactobacillus johnsonii CCFM1314 shows better regulation (down-regulated by 17.49% compared with the model group). Figure 3 (b) The results show that the expression of TLR-4 mRNA in the control group is 1, and the expression in the model group is 1.91, which is significantly increased compared with the control group. The expression of TLR-4 mRNA after the intervention of Lactobacillus delbrueckii DM8909, Lactobacillus johnsonii CCFM1314 and Lactobacillus gasseri QJSWX195M1 is 0.53, 0.61 and 0.60 respectively, and the probiotic intervention groups all show significant down-regulation. In summary, Lactobacillus johnsonii CCFM1314 can significantly weaken the inflammatory response of vaginal tissue.

[0084] (2) MAPK / AP-1 / MMP-1 mRNA expression in the skin tissue of the back of mice

[0085] Twenty mg of skin tissue was taken from the back of five groups of mice respectively, and the experimental steps were the same as in (1). The primer sequences are shown in Table 3.

[0086] Table 3 Primers and sequences

[0087]

[0088] Figure 4(a) The results show that the expression of MAPK mRNA in the control group is 1, and the expression in the model group is 5.61, which is significantly higher than that in the control group. The expression of MAPK mRNA after intervention of Lactobacillus delbrueckii DM8909, Lactobacillus johnsonii CCFM1314 and Lactobacillus gasseri QJSWX195M1 is 2.14, 4.60 and 0.98 respectively, which proves that the vaginal mice do have skin health risks, and oral administration of probiotics helps to resist such risks. Figure 4 (b) shows that the expression of AP-1 mRNA in the control group is 1, and the expression in the model group is 1.17, which is slightly higher than that in the control group. The expression of AP-1 mRNA after intervention of Lactobacillus delbrueckii DM8909, Lactobacillus johnsonii CCFM1314 and Lactobacillus gasseri QJSWX195M1 is 0.51, 0.28 and 1.41 respectively. Compared with other groups, Lactobacillus johnsonii CCFM1314 intervention group greatly reduces the expression of AP-1 mRNA, which is decreased by 76.07% compared with the model group, and the maximum decrease of other groups is 56.41%. Figure 4 (c) shows that the expression of MMP-1 mRNA in the control group is 1, and the expression in the model group is 1.34, which is significantly higher than that in the control group. The expression of MMP-1 mRNA after intervention of Lactobacillus delbrueckii DM8909, Lactobacillus johnsonii CCFM1314 and Lactobacillus gasseri QJSWX195M1 is 0.73, 0.98 and 0.86 respectively. All probiotic intervention groups significantly reduce the expression of MMP-1 mRNA (69.40% lower than the model group). In summary, Lactobacillus johnsonii CCFM1314 can significantly improve the skin aging problem caused by vaginitis.

[0089] (3) Col I and Col III content in the skin tissue of the back of the mice

[0090] Twenty mg of the skin tissue of the back of the mice in each of the five groups was taken respectively to detect the content of Col I and Col III, and the experimental steps were the same as those in (1).

[0091] The results are shown in Figure 5 (a), which shows the concentration of Col I in the skin tissue. The results show that compared with the control group (9.72 μg / L), the secretion of Col I in the vaginal tissue of the model group mice decreased significantly, with a concentration of 7.80 μg / L. Lactobacillus johnsonii CCFM1314 showed better regulation, and the concentration of Col I increased by 34.40% compared with the model group; and the maximum increase of Col I concentration in other groups was 30.36%. The results are shown in Figure 5(b), which shows the concentration of Col III in the skin tissue, and the results show that the Col III secreted by the vaginal tissue of the model group mice decreased significantly, with a concentration of 4.40 pg / L, as compared with the control group (8.14 pg / L). The concentration of Col III of L. johnsonii CCFM1314 increased by 22.95% as compared with the model group, while the concentration of Col III of L. delbrueckii DM8909 increased by only 5.22% as compared with the model group, and it can be seen that L. johnsonii CCFM1314 helps to generate skin collagen and resist skin aging of the mice with vaginitis.

[0092] (4) Colonization load of Gardnerella vaginalis in the vagina of the mice

[0093] A certain amount of phosphate buffer solution was sucked into a gun head to blow and suck the vagina of the five groups of mice back and forth to obtain 300 pL of vaginal lavage fluid samples, and the Gardnerella vaginalis and Lactobacillus were counted by qPCR. The whole experimental period was 17 days, and two samplings were performed during the period. The first sampling was on the 7th day (the first day after the infection ended) as the colonization test of Gardnerella vaginalis, and the second sampling was the colonization test after the intervention ended (the 17th day), and the load of Gardnerella vaginalis and Lactobacillus was quantified. First, according to the manufacturer's instructions, the soil fast DNA rotation kit (MP Biomedical, USA) and QIAQuick Gel extraction kit (Qiagen, Germany) were used to extract DNA in the vaginal lavage fluid. Subsequently, qPCR was used for quantitative detection of Gardnerella vaginalis and Lactobacillus. Primers were selected according to the 16S rRNA sequence of bacteria. The reaction mixture (10 pL) included 5 pL of 2x ChamQUniversal SYBR qPCR Master Mix (Nanjing Novozyme Biotech Co., Ltd.), 1 pL of template DNA (10 ng / pL), 0.5 pL of forward and reverse primers (10 pM each), and 3 pL 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, which was cycled for 40 times. Another step, 95°C for 10 s, from 65°C to 95°C, increasing by 0.5°C every 5 s, to establish the melting curve. The threshold cycle value (CT) was determined, and the copy number was calculated according to the standard curve (Log copies / pL vs. CT value). Each sample was checked in triplicate.

[0094] Table 4 Species-specific primers for real-time quantitative PCR detection of Gardnerella vaginalis

[0095]

[0096] From Figure 6The results showed that, except for the blank control group, approximately 12.43 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 CCFM1314 group was significantly reduced to 9.07 lg copies / μL. Compared with the model group, neither Lactobacillus delbrueckii DM8909 nor Lactobacillus gasseri QJSWX195M1 significantly reduced Gardnerella vaginalis colonization (p>0.05).

[0097] (5) Histopathological analysis of mouse vaginal tissue

[0098] 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).

[0099] HE staining of mouse vaginal tissue can effectively assess the inflammation status of vaginal tissue in different groups of mice. Figure 7 As shown, the vaginal epithelium in the blank control group was smooth and continuous, with an intact tissue structure and no obvious inflammatory cell infiltration. In the model group, the vaginal mucosal epithelium showed poor continuity, with superficial cell erosion forming pores, submucosal stroma congestion, and a large number of inflammatory cells infiltrating the epithelium and stroma. 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 johnsonii* CCFM1314 showed the opposite effect to *Lactobacillus gasseri* QJSWX195M1, for example, restoring epithelial continuity and reducing inflammatory cell infiltration. The recovery of mouse vaginal tissue after *Lactobacillus johnsonii* CCFM1314 intervention was superior to that of the *Lactobacillus delbrueckii* DM8909 group.

[0100] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the examples described above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus johnsonii ( Lactobacillus johnsonii Lactobacillus johnsonii, described in CCFM1314, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No:63710.

2. A microbial preparation comprising Lactobacillus johnsonii CCFM 1314 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of Lactobacillus johnsonii CCFM1314 in the microbial preparation is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

4. A pharmaceutical product comprising Lactobacillus johnsonii CCFM 1314 according to claim 1 or a microbial preparation according to claim 2 or 3.

5. The pharmaceutical product according to claim 4, characterized in that The content of the Lactobacillus johnsonii CCFM1314 in the pharmaceutical product is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

6. Use of Lactobacillus johnsonii CCFM 1314 according to claim 1 or a microbial preparation according to claim 2 or 3 in the manufacture of a pharmaceutical product for alleviating skin ageing caused by vaginitis.

7. Use according to claim 6, characterized in that, The effect of alleviating skin ageing caused by vaginitis includes increasing the amount of Col I and Col III in skin tissue.

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