CN112263596B - Application of bifidobacterium V9 in adhesion and bacteriostasis capacity of vaginal epithelial cells - Google Patents

Application of bifidobacterium V9 in adhesion and bacteriostasis capacity of vaginal epithelial cells Download PDF

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CN112263596B
CN112263596B CN202011125078.8A CN202011125078A CN112263596B CN 112263596 B CN112263596 B CN 112263596B CN 202011125078 A CN202011125078 A CN 202011125078A CN 112263596 B CN112263596 B CN 112263596B
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高杰
李翠平
马春红
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Inner Mongolia Puze Biologics Co ltd
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • A61K35/741Probiotics
    • A61K35/744Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745Bifidobacteria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10Antimycotics

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Abstract

The invention discloses application of bifidobacterium V9 in adhesion to vaginal epithelial cells and bacteriostasis capacity. The bifidobacterium V9 can strongly inhibit the adhesion of enterobacter and staphylococcus to vaginal epithelial cells, has high cure rate on vaginitis, obviously improves the inflammatory reaction of uterus, reduces the phenomena of tissue congestion and edema, reduces the expression of inflammatory factors IL-1 beta and TNF-alpha in rat serum, improves the expression of an anti-inflammatory factor IL-4, and shows that the bifidobacterium V9 has a positive effect on the rehabilitation of the rat vaginitis, thereby laying a foundation for developing a novel microecological preparation for treating the vaginal inflammation.

Description

Application of bifidobacterium V9 in adhesion and bacteriostasis capacity of vaginal epithelial cells
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to application of bifidobacterium to adhesion and bacteriostatic ability of vaginal epithelial cells.
Background
Human health is fundamentally greatly influenced by the microbial ecosystem of the organism, and the microbial ecosystem of the reproductive tract of women is characterized in that bacterial communities with different relative proportions change to mark health state change. The female genital tract is a very complex micro-ecosystem, in which lactic acid bacteria, as the dominant micro-organism, play a major role in maintaining the vaginal micro-ecological environment balance. When the probiotics in the vagina are reduced or disappeared, other pathogenic microorganisms can grow rapidly, so that bacterial vaginitis, fungal vaginitis and sexually transmitted diseases can be caused.
Bacterial vaginitis is a main infectious disease of a gynecological reproductive system, the morbidity of the bacterial vaginitis is 10% -41%, and at present, the bacterial vaginitis is mainly treated by adopting an antibiotic therapy, so that the micro-ecological environment of the reproductive system is disordered, secondary candida vaginosis is caused, and the problems of poor treatment effect, high recurrence rate and the like are caused. The health state of human body is closely related to intestinal flora, and the research is of far-reaching significance for developing a novel microecological preparation for treating vagina inflammation.
Disclosure of Invention
According to one aspect of the present invention, there is provided the use of bifidobacterium V9 for its ability to adhere to and inhibit bacterial growth in vaginal epithelial cells. The bifidobacterium V9 is self-produced bacterial powder of the applicant, and the bifidobacterium V9 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and is classified and named as follows: bifidobacterium animalis with the preservation number of CGMCC No.4473, the preservation date of 2010.12.14 and the preservation address: west road No. 1, north chen, township, beijing, institute for microbiology, china, depository: china Committee for culture Collection of microorganisms general culture Collection.
The antibacterial mechanism of bifidobacterium V9 relates to various metabolites produced by it, including lipoteichoic acid, peptidoglycan, acidic substances, lactein, carbon dioxide, hydrogen peroxide, and the like. Wherein the lipoteichoic acid and peptidoglycan have adhesion effect on epithelial cells, and acidic substances can consume a large amount of cell energy and influence the stability of cell membranes; lactein acts on the cell membrane, causing leakage of substances and energy within the membrane.
The application detects the average levels of the cell inflammatory factors IL-1 beta, TNF-alpha and IL-4, researches the action of bifidobacterium V9 in the expression of the cell inflammatory factors and the action mechanism of bifidobacterium V9 in the vaginal bacteriostasis, and provides a basis for developing a microecological preparation.
The bifidobacterium V9 of the invention has high viable count and strong adhesion, and is supported by a large amount of experimental data and proved by clinical tests, and toxicological experiments and safety evaluations are carried out. And the animal experiment data are obvious. Compared with the commercial probiotics, the probiotic bacteria have high cost performance, are more substantial, and have obvious effect of treating vaginitis.
The result of the study by comparing the adhesion of the bifidobacterium with the adhesion of the enterobacter, the staphylococcus and the candida albicans to vaginal epithelial cells shows that the bifidobacterium V9 is obviously higher than the adhesion of the enterobacter, the staphylococcus and the candida albicans; the bifidobacterium V9 can strongly inhibit the adhesion of enterobacter, staphylococcus and candida albicans to vaginal epithelial cells. The space occupation formed by the adhesion of bifidobacterium V9 is an important protective mechanism to prevent the attachment of other bacteria to vaginal tissue.
The artificial rat vaginitis model, by taking metronidazole as a control and using bifidobacterium V9 for intervention treatment, results show that the negative conversion rate of the bifidobacterium V9 group is 70%, the cure rate of mixed bacteria vaginitis of rats is 62.1, the inflammatory reaction of uterus is obviously improved, and the phenomena of tissue congestion and edema are reduced. Bifidobacterium V9 reduces the expression of inflammatory factors IL-1 beta and TNF-alpha in rat serum and increases the expression of anti-inflammatory factor IL-4. Shows that the bifidobacterium V9 has a positive effect on the recovery of rat vaginitis.
Drawings
FIG. 1 is a graph showing the adhesion of Bifidobacterium V9 of the present invention to vaginal epithelial cells;
FIG. 2 shows rat uterus and slices;
FIG. 3 shows the expression of IL-4, a cytokine in rat serum;
FIG. 4 shows the expression of the cytokine IL-1. beta. in rat serum;
FIG. 5 shows the expression of the inflammatory cytokine TNF-. alpha.in rat serum.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Experiment 1 adhesion of Bifidobacterium V9 to vaginal epithelial cells
Vaginal epithelial cells:
the vaginal suppository is taken from the vagina of 17 healthy women, the subjects are 19-40 years old, the gynecological examination is normal, and the asexual life history is within 3 days. The scraped mucosal epithelial cells were immediately suspended in 10ml of RPMI-1640, the cells were washed 3 times with shaking in a vortex mixer, and endogenous bacteria were removed by suction filtration through a glass filter. Gram staining, microscopic examination to determine whether all endogenous bacteria were removed. The plates were counted and the cell concentration was adjusted to 1X 105/ml.
Bacteria:
the bifidobacterium V9 strain is separated and preserved by the company of limited liability of biological products of inner Mongolia Puze; the enterobacteria, staphylococcus and candida albicans are all isolated from the vagina of a patient with vaginitis. Respectively inoculating the bacteria to LBS, EMB.SP and SB solid culture media, and culturing at 37 deg.C for 18 h; before adhesion analysis, lawn was scraped and bacteria were washed 3 times (2000rpm 10min) in RPMI-1640 adjusted to a bacterial concentration of 1X 108/m 1.
Adhesion analysis:
mixing vaginal epithelial cell suspension and bacterial suspension 1ml each, incubating at 37 deg.C for 2 hr, washing cells with RPMI-1640 for 3 times, and filtering with glass filter to remove non-adhered bacteria. Smearing, drying at room temperature, fixing with methanol for 10min, gram staining, and observing under a microscope.
1.4 Effect of Bifidobacterium V9 on the adhesive Capacity of Enterobacter, Staphylococcus, Candida albicans
Mixing Bacillus bifidus V9 bacterial suspension 1m1 and vaginal epithelial cell suspension 1m1, incubating at 37 deg.C for 30min, centrifuging (1500rpmX 5min), discarding supernatant 1ml, adding 1ml of Enterobacter, Staphylococcus, and Candida albicans respectively, mixing, and incubating at 37 deg.C for 2 h. pH 4.5.
Experiment 2 animal experiment vaginitis model establishment and treatment
Establishing a rat vaginitis model:
the experimental groups comprise a negative control group, a model group, metronidazole treatment and a bifidobacterium V9 group, wherein each group comprises 20 animals;
antibiotic treatment, cutting medical absorbable gelatin sponge into 0.5cmx0.5cm, and sucking 50 muL of antibiotic with the concentration of 100 mg/mL; after the gelatin is fully saturated by antibiotics, wearing a bite-preventing glove, grasping the back skin of the rat by the left hand and fixing the head, and holding the rat tail by the right hand and inverting the rat; another person inserts gelatin sponge containing antibiotics and fills the gelatin sponge into the vagina and the uterine cavity; antibiotic treatment is carried out for 6 times, and recovery is carried out for three days continuously;
after antibiotic treatment, preparing a mixed bacterial solution with the concentration of 3x1010CFU/mL by using type B hemolytic streptococcus, staphylococcus aureus and large intestine according to the proportion of 1:1: 2; cutting medical absorbable gelatin sponge into size of 0.5cmx0.5cm, and sucking 50 μ L to make the gelatin sponge fully absorb; after the gelatin fully absorbs the mixed bacterial solution and is saturated, wearing a bite-preventing glove, grasping the back skin of the rat by the left hand and fixing the head, and holding the rat tail by the right hand and inverting the rat; another person clamps gelatin sponge containing mixed bacteria solution, and fills into vagina and uterine cavity, and stands upside down for 1-2 min; blank group is PBS treatment; and (4) molding for 7 times, stopping the medicine for one day after three days, and stopping the medicine for one day after molding.
Treatment of rat vaginitis model:
7 times of administration treatment are carried out after the molding is successful. The administration dosage of the metronidazole treatment group is obtained according to the purchased metronidazole medicine instruction and equivalent dosage conversion, and the administration dosage of the rat is 16.2mg/200 g; a0.32 g/mL solution of metronidazole 81mg dissolved in sterile PBS 250 μ L was administered at a dose of 16.2mg/200g per rat. Cutting medical absorbable gelatin sponge into size of 0.5cmx0.5cm, sucking 50 μ L of medicine to make gelatin sponge fully absorb the medicine, and placing into vagina of rat; after the evaluation of the viable count of the freeze-dried powder, weighing 0.3g of bacterial powder, and preparing 1x1011CFU/mL of mixed bacterial liquid by using sterile PBS; shearing medical absorbable gelatin sponge into a size of 0.5cmx0.5cm, sucking 50 mu L of bacterial liquid, putting the gelatin sponge into a vagina of a rat after the gelatin sponge fully absorbs the bacterial liquid, wherein each experimental rat is 5x109 CFU;
experimental rats were sacrificed by cervical dislocation. Dissecting a rat, taking uterus tissue, storing part of the uterus tissue in 4% paraformaldehyde solution, performing ELISA and HE staining detection on part of the uterus tissue, and directly storing part of the uterus tissue in-80 refrigerator for later use.
Experiment 3 detection of various indexes after treatment of animal experiment vaginitis model
(1) Negative conversion rate of mixed bacterial infection
After stopping drug administration, taking vaginal secretion of rats at 1d, 2d and 5d for identification, identifying generation of sterile filaments in vaginal lavage fluid of rats and determining that colony count is less than or equal to 10 as negative turning.
(2) Degree of inflammation and cure rate of vaginal affection
On day 7 of drug withdrawal, the rats were sacrificed for dissection and vaginal specimens were taken for histopathological examination. The vagina specimen is graded according to 4 basic indexes of congestion, edema, hemorrhage and infiltration, the sum of the inflammation grading values of each index is the total inflammation grading value, the grading value of the model group is 100 percent of the inflammation degree, the inflammation degree of the administration group can be obtained by comparing the administration group with the model group, and the inflammation degree of the drug group subtracted by 100 percent is the cure degree of the vaginitis (100 percent to the inflammation degree of the drug group).
TABLE 1 evaluation criteria for inflammatory irritation of vaginal mucosa in rats
Figure RE-GDA0002811097570000041
(3) HE staining of rat uterine tissue
And (3) dehydrating: taking out fixed uterus, washing with PBS, dehydrating with ethanol, and dehydrating with 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and 95% ethanol for 15 min; dehydrating with anhydrous ethanol twice for 60 min; .
Embedding: treating the dehydrated tissue with xylene for 30min, and embedding with paraffin;
slicing and sealing: cutting a flat plane by a rotary machine, setting the thickness of the cut plane to be 55 mu m, carefully picking up the cut wax sheets, placing the wax sheets in a water tank of a sheet spreading machine with the water temperature of 45 ℃, and spreading and evaluating the wrinkled wax sheets; and (3) placing the wax sheet on a glass slide, placing the glass slide on a 60 ℃ baking machine, and baking for 4 hours.
Dewaxing: dewaxing with xylene (I) and (II) for 15min, respectively, treating with xylene (anhydrous ethanol) 1:1 solution for 5min, and treating with ethanol solution with decreasing concentration, 100% ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 5 min.
Dyeing: staining Harris hematoxylin for 5min, and treating with ethanol solution with decreasing concentration, 70 % ethanol 5, 80% ethanol, 95% ethanol, and 100% ethanol for 5 min; staining with 0.5% eosin for 1 min; sequentially treating with 100% ethanol (I) and 100% ethanol (II) for 5 min; adding xylene (I) and xylene (II) respectively, and allowing to stand for 10 min; sealing neutral gum into a sheet;
and (4) observation: the mounting pieces are placed under an optical microscope and photographed and observed under a 200-fold mirror and a 400-fold mirror respectively, and are analyzed for pathophysiology.
(4) Determination of cytokines IL-1 beta, TNF-alpha, IL-4
The method comprises the following steps of collecting about 3mL of whole blood by cardiac puncture, collecting blood samples by using blood collection tubes of corresponding groups, standing for 1h at room temperature, enabling the blood to be coagulated, separating out serum, using a pipette to transfer the upper serum, sucking the serum into a new centrifugal tube, placing the centrifugal tube containing the serum into a high-speed centrifuge with the temperature of 4 ℃ and the rmp of 3000, centrifuging for 15min, and immediately placing the centrifugal tube into a refrigerator with the temperature of 80 ℃ below zero for freezing for later use. The average levels of the cytokines IL-1 beta, TNF-alpha and IL-4 in the serum of each group of rats were determined by performing the procedures strictly according to the instructions of the ELISA kit.
Results and analysis of experiment 4
(1) The adhesion of bifidobacterium V9 to vaginal epithelial cells is high in viable count and strong in adhesion, as shown in figure 1, of bifidobacterium V9.
(2) Adhesion of bacteria to vaginal epithelial cells
As can be seen from Table 2, at pH4.5, Bifidobacterium V9 has significantly higher adhesion to vaginal epithelial cells than Enterobacter, Staphylococcus, and Candida albicans.
TABLE 2 vaginal epithelial cell adhesion bacteria count
Figure RE-GDA0002811097570000051
(3) Negative conversion rate of mixed bacteria infection
As shown in Table 3, the vaginal secretion microscopic examination of the animals in the blank control group is negative, and compared with the control group of the model, the difference has statistical significance (P is less than 0.01), which shows that the model construction of the rat model with the mixed bacterial vaginitis is successful, the negative conversion rate of the bifidobacterium V9 is 70%, and the difference is obvious compared with the model group, which shows that the bifidobacterium V9 has positive effect on the rat with the vaginitis.
TABLE 3 negative conversion rate of mixed bacteria infection
Figure RE-GDA0002811097570000052
Figure RE-GDA0002811097570000061
(4) Degree of inflammation and cure rate of vaginal affection
From the table 4, the cure rate of the rat mixed bacteria vaginitis of the bifidobacterium V9 dose group and the metronidazole group is 62.1 and 93.1, and compared with the model control group, the difference is significant (P is less than 0.05).
TABLE 4 degree of inflammation and cure rate of vaginal affection
Figure RE-GDA0002811097570000062
(5) HE staining of rat uterine tissue
As shown in figure 2, the uterus morphology of each group of rats, the blank group, the uterus morphology structure and the size of the rats are normal, and serosa has no congestion and edema phenomenon; the model group comprises rat uterine serosa with serious congestion, thinned uterine wall and lumen, clear liquid can be seen in the uterine cavity, and most of uterine cavities have tortuous swelling; the metronidazole group is characterized in that the inflammatory hyperemia of the uterus of the rat is basically disappeared without edema phenomenon; bifidobacterium V9, atrophy and deformation of uterine cavity.
As shown in figure 2, HE staining of uterine tissues of rats in the blank group shows that the morphological structure of endometrial goblet cells in the convalescent period is complete and is orderly arranged, cell nuclei are positioned at the inner side of vaginal mucosa goblet cells, and the phenomena of uterine congestion, edema and lymphocyte infiltration are avoided; the uterine tissue in the convalescent model group is seriously congested and is accompanied with inflammatory cell infiltration phenomenon, and the integrity of vaginal mucous epithelial cells is damaged; the metronidazole group has the advantages that the whole goblet cells of the uterine wall chorion are orderly arranged, the tissue congestion and edema completely disappear, and the inflammatory response of the uterus of the rat is obviously improved. After recovery period of bifidobacterium V9 group, vaginal mucosal epithelial cells are still shed, and goblet cells are not regularly arranged.
(6) Expression of cytokines IL-1 beta, TNF-alpha, IL-4
As shown in fig. 3-5, the content of IL-4 in the serum of the rat in the vaginitis model group is significantly lower than that in the blank group (P < 0.01), wherein the content of IL-4 in the serum of the metronidazole group and the bifidobacterium V9 group is significantly different from that of the model group (P < 0.05); the content of IL-4 in the serum of the rats in the metronidazole group is slightly higher than that in the bifidobacterium V9 group. The model reduces the IL-4 content in the serum of the rat in the model group, and after the treatment by metronidazole and bifidobacterium V9, the IL-4 content in the serum of the rat is effectively improved. The content of IL-1 beta in the blood serum of a rat in the vaginitis model group is extremely obviously higher than that of a blank group, wherein the content of IL-1 beta in the blood serum of the metronidazole group and the bifidobacterium V9 group is obviously different from that of the model group; the content of IL-1 beta in the serum of the rats in the metronidazole group is slightly lower than that in the bifidobacterium V9 group. The IL-1 beta content in the blood serum of the rats in the model group is increased by modeling, and the IL-1 beta content of inflammatory factors in the blood serum of the rats is effectively reduced after the treatment by metronidazole and bifidobacterium V9. The content of TNF-alpha in the serum of a rat in the vaginitis model group is extremely obviously higher than that of a blank group, wherein the content of TNF-alpha in the serum of the metronidazole group and the bifidobacterium V9 group is obviously different from that of the model group; the content of TNF-alpha in the serum of the rats in the metronidazole group is slightly lower than that in the bifidobacterium V9 group. The content of TNF-alpha in the serum of the rat of the model group is increased by modeling, and the content of inflammatory factor TNF-alpha in the serum of the rat is effectively reduced after the metronidazole and bifidobacterium V9 are used for treatment.
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

Claims (1)

1. Application of a medicament containing Bifidobacterium V9 in preparing a product for reducing increase of serum cytokine IL-1 beta caused by vaginitis of an organism, wherein the Bifidobacterium V9 is Bifidobacterium animalis (CGMCC number 4473).
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