Lactobacillus salivarius and its use in the preparation of a medicament for treating emt

Abstract

The present application relates to the field of biological medical technology, and in particular to saliva combined lactobacillus and its application in preparing a drug for treating EMT. The saliva combined lactobacillus NCU-03 has a probiotic property, can reduce the production of blood vessels and the proliferation of cells of endometrial ectopic lesions by inhibiting the expression of VEGF and PCNA, and can inhibit the development of endometrial ectopic by reducing the expression level of IL-6, IL-1beta and TNF-alpha. Therefore, the saliva combined lactobacillus NCU-03 can be used for treating endometrial ectopic.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to *Lactobacillus saliva-associated* and its application in the preparation of drugs for treating EMT. Background Technology

[0002] Endometriosis (EMT) is an estrogen-dependent, chronic inflammatory, hereditary disease characterized by the extrauterine displacement of functional endometrial tissue. Clinical manifestations include progressive pelvic pain, infertility, and systemic inflammatory response. Current diagnosis relies on surgical pathological examination. The pathogenesis involves multiple factors, including retrograde implantation and coelomic metaplasia, but a unified molecular mechanism to explain clinical heterogeneity is lacking.

[0003] Currently, treatment for EMT primarily involves medication (oral contraceptives, progestins, GnRH agonists, etc.) and surgery. However, medications suffer from tolerability issues, and conservative surgery has a high recurrence rate. Therefore, there is an urgent need to develop new treatment strategies with low recurrence rates and low side effects. Summary of the Invention

[0004] In view of this, the present invention provides *Lactobacillus saliva-associated* and its application in the preparation of medicaments for treating EMT, thereby at least solving one problem existing in the prior art.

[0005] In a first aspect, the present invention provides a strain of *Lactobacillus salivarius* (… Ligilactobacillus salivarius NCU-03, the aforementioned Lactobacillus salivae NCU-03 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 8, 2022, with accession number CGMCC No. 25505.

[0006] The 16S rRNA sequencing results of the aforementioned *Lactobacillus saliva-associated* NCU-03 are shown in SEQ ID No. 1, which is detailed below:

[0007]

[0008] Lactobacillus salivarius NCU-03 possesses probiotic properties, reducing angiogenesis and cell proliferation in endometriotic lesions by inhibiting VEGF and PCNA expression, and suppressing the development of endometriosis by lowering the expression levels of IL-6, IL-1β, and TNF-α. Therefore, Lactobacillus salivarius NCU-03 can be used to treat endometriosis.

[0009] Secondly, the present invention provides the use of the above-mentioned Lactobacillus saliva-associated NCU-03 in the preparation of a medicament for the prevention or treatment of endometriosis.

[0010] Thirdly, the present invention provides a medicament for the prevention or treatment of endometriosis, comprising the aforementioned Lactobacillus saliva-associated with NCU-03.

[0011] In some optional embodiments, the dosage form of the above-mentioned drug for preventing or treating endometriosis is a liquid formulation.

[0012] In some alternative embodiments, the aforementioned medicaments for the prevention or treatment of endometriosis may also include pharmaceutically acceptable excipients.

[0013] In some alternative embodiments, the pharmaceutically acceptable excipients mentioned above are water, saline, or gelatin saline.

[0014] In some optional embodiments, the concentration of the above-mentioned saliva-associated lactobacillus NCU-03 is 0.1 × 10⁻⁶. 9 CFU / mL - 10 × 10 9 CFU / mL. Preferably, the concentration of the above-mentioned saliva-associated lactobacillus NCU-03 is 1×10⁻⁶ CFU / mL. 9 CFU / mL - 5 × 10 9 CFU / mL. Most preferably, the concentration of the above-mentioned saliva-associated lactobacillus NCU-03 is 1×10⁻⁶ CFU / mL. 9 CFU / mL.

[0015] Due to the adoption of the above technical solutions, the embodiments of the present invention have the following beneficial effects: a strain of Lactobacillus salivarius NCU-03 is provided, which has prebiotic properties and can be used to prepare a drug for treating endometriosis. Attached Figure Description

[0016] Figure 1 This is a growth curve of Lactobacillus saliva-associated with NCU-03 in an embodiment of the present invention.

[0017] Figure 2 This is a statistical chart showing the acid resistance test results of Lactobacillus saliva-associated NCU-03 in the embodiments of the present invention.

[0018] Figure 3 This is a statistical chart showing the results of the bile salt tolerance test of Lactobacillus NCU-03 in saliva in an embodiment of the present invention.

[0019] Figure 4 This is a graph showing the results of the antagonistic activity assay of the pathogen Lactobacillus NCU-03 in saliva in an embodiment of the present invention.

[0020] Figure 5 This is a statistical chart showing the results of the antagonistic activity assay of the pathogen Lactobacillus NCU-03 in saliva in this embodiment of the invention.

[0021] Figure 6 This is a graph showing the results of an antibiotic resistance test on Lactobacillus saliva-associated NCU-03 in an embodiment of the present invention.

[0022] Figure 7 This is a statistical chart showing the results of antibiotic resistance experiments on Lactobacillus saliva-associated NCU-03 in this embodiment of the invention.

[0023] Figure 8 These are photographs of endometriotic lesions in mice in various groups according to embodiments of the present invention.

[0024] Figure 9 This is a statistical diagram of the volume of endometriotic lesions in each group of mice in the embodiments of the present invention.

[0025] Figure 10 This is a statistical chart showing the weight of endometriotic lesions in each group of mice in this embodiment of the invention.

[0026] Figure 11 The images show the HE staining and immunohistochemical PCNA and VEGF staining results of endometriotic lesions in mice in each group of embodiments of the present invention.

[0027] Figure 12 This is a statistical chart showing the expression levels of IL-6, IL-1β, and TNF-α in the peritoneal fluid of mice in each group in this embodiment of the invention. Detailed Implementation

[0028] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully illustrate the purpose, solution and effects of the present invention.

[0029] Isolation and identification of Lactobacillus saliva-associated with NCU-03:

[0030] Fecal samples were collected from centenarian patients at Nanchang Central Hospital and transferred to centenarian tubes containing 5 mL of PBS solution. The samples were resuspended using a shaker, thoroughly mixed, and the supernatant was serially diluted 10⁻⁶ times. 0 -10 7The culture was plated. From plates containing 100-300 colonies, 5-10 single colonies were inoculated into 5 mL of MRS liquid medium and cultured aerobically and anaerobicly for 24-28 h. The activated bacterial culture was preserved (one tube per colony). The remaining culture was centrifuged, and the DNA was extracted for high-throughput sequencing. The sequencing result of one colony, shown in SEQ ID No. 1, confirmed it to be *Lactobacillus salivarius* (…). Ligilactobacillus salivarius It was named Lactobacillus salivarii ( Ligilactobacillus salivarius NCU-03 is deposited at the China General Microbiological Culture Collection Center of the China Microbiological Culture Collection Committee.

[0031] Evaluation of the probiotic properties of Lactobacillus salivarius NCU-03:

[0032] Lactobacillus saliva-associated NCU-03 was inoculated into MRS liquid medium and cultured in a shaker at 37°C. The OD value of the bacterial culture at 600 nm was measured every 2 hours. 600nm The result is as follows: Figure 1 As shown, Lactobacillus salivarius NCU-03 entered the logarithmic growth phase after 4 hours and reached the stationary phase after 12 hours.

[0033] Saliva-associated Lactobacillus NCU-03 was resuscitated in MRS liquid medium and cultured overnight at 37°C with a shaker for 24 h, with activation twice. One mL of bacterial culture was taken, centrifuged (6000 rpm for 4 minutes) to remove the supernatant, and the remaining cells were washed with PBS to remove the medium. The cells were then resuspended in phosphate-buffered saline (PBS) at different pH values ​​(1.0, 3.0, 5.0, 7.0) and incubated at 37°C for 4 hours. Subsequently, the viable cell count was calculated under each pH condition using a plate count method. Results are as follows: Figure 2 As shown, *Lactobacillus salivarius* NCU-03 can survive in gastric acid; under acidic conditions of pH 1, the viable count of *Lactobacillus salivarius* NCU-03 remains at 10. 4 CFU / mL level.

[0034] Saliva-associated Lactobacillus NCU-03 was revived using MRS liquid medium and cultured overnight at 37°C with a shaker for 24 h, followed by two activation cycles. One mL of bacterial culture was collected, centrifuged (6000 rpm for 4 min) to remove the supernatant, and the remaining cells were washed with PBS to remove the medium. The cells were then resuspended in phosphate-buffered saline (PBS) at different bile salt concentrations (0%, 0.1%, 0.2%, 0.3%) and incubated at 37°C for 4 h. The viable cell count under each condition was then calculated using a plate count method. Results are as follows: Figure 3As shown, *Lactobacillus salivarius* NCU-03 exhibits concentration-dependent tolerance to bile salts; even at a bile salt concentration of 0.3%, the viable count can reach 10-1. 5 CFU / mL.

[0035] Using soybean broth (LB) liquid medium for Staph.aureus Cowan1, C. albicans SC531 E. coli O157、 Shigella flexneri ATCC 12022 Salmonella typhimurium ATCC 13311 Pseudomonas aeruginosa , L. monocytogenes , Beta hemolytic streptococcus The pathogenic bacteria were cultured, and after 24 hours, the bacterial suspension was evenly spread onto the surface of LB agar medium. Oxford cups were then placed in the cups, and 250 μL of the culture supernatant of the cultured probiotics was added (probiotic group); the control group used MRS medium. After 6-8 hours of observation, the size of the inhibition zone (zone of growth inhibition) was measured. Results are as follows: Figure 4 and Figure 5 As shown, saliva-associated Lactobacillus NCU-03 inhibits Staph.aureus Cowan1, C. albicans SC531 E. coli O157、 Shigella flexneri ATCC12022 Salmonella typhimurium ATCC 13311 Pseudomonas aeruginosa , L. monocytogenes , Beta hemolytic streptococcus (These pathogens in) Figure 4 The numbers are 1 to 8 in sequence. Figure 4 In the text, 'a' represents the control group. Figure 4 The average diameters of the probiotic groups (b represents the probiotic group) were 20.7 mm, 21.0 mm, 19.0 mm, 19.3 mm, 20.7 mm, 19.7 mm, 19.3 mm and 19.2 mm, respectively.

[0036] Take 30 μL of activated saliva-based Lactobacillus NCU-03 bacterial suspension and spread it evenly on the surface of an agar plate. Then, place antimicrobial susceptibility test discs (erythromycin, penicillin, tetracycline, chloramphenicol, ceftriaxone, ampicillin, ciprofloxacin CIP, lincomycin MY) on the agar plate surface. Incubate the plates at 37℃ for 16 to 18 hours. After incubation, measure and record the diameter of the inhibition zone. Results are as follows: Figure 6 and Figure 7 As shown, Lactobacillus saliva-associated NCU-03 exhibits high sensitivity to erythromycin (E), penicillin (PEN), tetracycline (TET), chloramphenicol (C), ceftriaxone (CTR), and ampicillin (AMP).

[0037] These results indicate that Lactobacillus saliva-associated with NCU-03 possesses favorable probiotic properties.

[0038] Evaluation of the effect of Lactobacillus salivarius NCU-03 on endometriosis:

[0039] Animal experiments were conducted using 8-week-old female BALB / c mice to establish an endometriosis model via allogeneic endometrial transplantation for evaluation. Mice were housed in a specific pathogen-free (SPF) laboratory, weighing 18-22 grams. One week prior to surgery, donor mice were subcutaneously injected with 3 μg of estradiol benzoate to induce estrus, resulting in a hydrocele-filled uterus. Mice were randomly divided into a control group (C group), an endometriosis model group (M group), and a probiotic treatment group (MP group), with 6 mice in each group. Endometriosis models were established in the M and MP groups according to the following steps:

[0040] (1) After the donor mice were anesthetized with isoflurane, they were euthanized by cervical dislocation, fixed in a supine position, the abdomen was prepared and disinfected with povidone-iodine, a sterile surgical drape was laid, sterile gloves were put on, and the abdominal cavity was entered layer by layer along the midline of the abdomen.

[0041] (2) Locate the Y-shaped bicornuate uterine structure of the mouse, gently pull the bilateral uterine horns to the surgical field, and separate the mesentery from the surrounding connective tissue;

[0042] (3) The junction of the uterine horn and the ovary is severed, and a transverse cut is made in the Y-shaped isthmus of the uterus to obtain both uteri intact. The uteri are placed in sterile saline and repeatedly rinsed and separated for further separation.

[0043] (4) Place the uterine horn in a sterile curved tray, use ophthalmic scissors to longitudinally cut open the uterus to expose the endometrium, and trim it into endometrial pieces of about 3 mm × 3 mm in size;

[0044] (5) The tissue block was sutured to the parietal peritoneum of the recipient mouse using 6-0 sutures, and the abdomen was closed layer by layer and then disinfected with povidone-iodine.

[0045] Mice in group C were given parietal peritoneal sutures. After model establishment, recipient mice were subcutaneously injected with 3 μg of estradiol benzoate to promote the establishment of the EMT mouse model.

[0046] The day the endometriosis model was established was designated as day 0. On day 21, the mice in each group were treated as follows:

[0047] Group C: Control mice were administered 0.1 ml of physiological saline by gavage every two days for a total of 3 weeks;

[0048] Group M: Mice with endometriosis model were administered 0.1 ml of physiological saline by gavage every two days for a total of 3 weeks;

[0049] MP group: Mice with endometriosis were administered 0.1 ml of Lactobacillus saliva-containing NCU-03 bacterial suspension by gavage every two days for 3 weeks. The Lactobacillus saliva-containing NCU-03 bacterial suspension was obtained by resuspending two generations of revived Lactobacillus saliva-containing NCU-03 bacteria in gelatin saline, with a concentration of 1×10⁻⁶. 9 CFU / mL.

[0050] On the morning of the second day after drug administration, mouse feces were collected uniformly, and changes in fecal microbiota were detected by qPCR. Mice were sacrificed on day 42, and blood, peritoneal fluid, and ectopic lesion tissue were collected for further analysis.

[0051] like Figures 8-10 As shown, the volume of ectopic lesions in group M mice was 238.90 ± 44.34 mm. 3 The volume of ectopic lesions in the MP group mice was 118.10 ± 17.45 mm. 3 That is, the volume of ectopic lesions in the MP group mice was significantly reduced. p <0.01). The weight of ectopic lesions in group M mice was 0.300±0.035 g, while that in group MP mice was 0.219±0.023 g, indicating that the weight of ectopic lesions in group MP mice was also lighter (p<0.05). This demonstrates that *Lactobacillus salivarius* NCU-03 can reduce the volume and weight of endometriotic lesions in mice.

[0052] Figure 11 HE staining of ectopic lesions in mice showed that the ectopic lesions in group M mice contained glandular tissue and stromal cells, consistent with the pathological characteristics of endometriosis. The endometriotic lesions in group MP mice all had smaller stromal areas and fewer glands. Figure 11 Immunohistochemical results showed that, compared with the M group, the MP group had decreased expression of VEGF and PCNA in endometriotic lesions. These results indicate that *Lactobacillus salivarius* NCU-03 can reduce angiogenesis and cell proliferation in endometriotic lesions, thereby delaying the progression of endometriosis.

[0053] The levels of IL-6, IL-1β, and TNF-α in the peritoneal fluid of mice were detected by ELISA. The results are as follows: Figure 12As shown, compared with the M group mice, the MP group mice had reduced expression levels of IL-6, IL-1β, and TNF-α in their peritoneal fluid. IL-6, IL-1β, and TNF-α are common pro-inflammatory cytokines, and these inflammatory factors play an important role in promoting the progression of endometriosis. The combination of saliva and Lactobacillus NCU-03 can reduce the levels of these inflammatory factors, which may help inhibit the development of endometriosis.

[0054] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention using the same means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. A strain of *Lactobacillus salivarius* ( Ligilactobacillus salivarius NCU-03, characterized in that, The Lactobacillus salivae NCU-03 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on August 8, 2022, with accession number CGMCC No. 25505.

2. The use of the Lactobacillus saliva-associated NCU-03 according to claim 1 in the preparation of a medicament for the prevention or treatment of endometriosis.

3. A drug for the prevention or treatment of endometriosis, characterized in that, Includes the Lactobacillus salivans NCU-03 as described in claim 1.

4. The medicament for preventing or treating endometriosis according to claim 3, characterized in that, The medication for preventing or treating endometriosis is in liquid form.

5. The medicament for preventing or treating endometriosis according to claim 3, characterized in that, The medications for the prevention or treatment of endometriosis also include pharmaceutically acceptable excipients.

6. The medicament for preventing or treating endometriosis according to claim 5, characterized in that, The pharmaceutically acceptable excipients are water, physiological saline, or gelatin physiological saline.

7. The medicament for preventing or treating endometriosis according to claim 3, characterized in that, The concentration of the saliva-associated lactobacillus NCU-03 was 0.1 × 10⁻⁶. 9 CFU / mL - 10×10 9 CFU / mL.

8. The medicament for preventing or treating endometriosis according to claim 7, characterized in that, The concentration of the saliva-associated lactobacillus NCU-03 was 1×10⁻⁶. 9 CFU / mL - 5 × 10 9 CFU / mL.

9. The medicament for preventing or treating endometriosis according to claim 8, characterized in that, The concentration of the saliva-associated lactobacillus NCU-03 was 1×10⁻⁶. 9 CFU / mL.

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