Lactobacillus crispatus and application thereof in preparation of vulva lichen sclerosus related drugs

Abstract

The invention relates to the technical field of biomedical treatment, in particular to lactobacillus crispatus and application thereof in preparation of vulva lichen sclerosus related drugs. In the invention, a strain of lactobacillus crispatus NCU-31 is provided, and the lactobacillus crispatus NCU-31 has an inhibition effect on a plurality of common pathogenic bacteria on a human body and has a relatively strong antioxidant effect; the metagen of the lactobacillus crispatus NCU-31 can be used for treating vulva lichen sclerosus, and is good in treatment effect, safe and free of side effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to Lactobacillus crispatus and its application in the preparation of drugs related to vulvar lichen sclerosus. Background Art

[0002] Vulvar lichen sclerosus (VLS) is a chronic vulvar skin lesion, mainly characterized by atrophy of the vulvar and perianal skin and mucosa. The course of the disease is slow and it is prone to relapse. If not treated promptly and regularly, it may lead to vulvar atrophy, adhesion, scarring, and even anatomical deformity and loss of function, and the risk of canceration will also increase. The disease is relatively easy to diagnose but difficult to treat. Early detection and intervention can effectively control the development of the disease and improve the prognosis.

[0003] Currently, the treatment of VLS mainly relies on local medication, and glucocorticoids (such as clobetasol propionate) or calcineurin inhibitors (such as tacrolimus) can be used. Physical therapies such as focused ultrasound or phototherapy can also assist in improving symptoms, and surgical intervention is required for severe adhesions or deformities.

[0004] However, the existing drugs for treating VLS may cause local irritation, burning sensation, dryness, or produce side effects, and are prone to relapse after drug withdrawal. Summary of the Invention

[0005] In view of this, the present invention provides Lactobacillus crispatus and its application in the preparation of drugs related to vulvar lichen sclerosus, which solves at least one problem existing in the prior art.

[0006] In a first aspect, the present invention provides a strain of Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31. The above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 has been deposited in the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being August 29, 2024, and the deposit number being CGMCC NO. 31777.

[0007] Among them, the full-length sequencing result of the 16sRNA of the above-mentioned Lactobacillus crispatus NCU-31 is shown in SEQ ID No.1, and SEQ ID No.1 is specifically as follows:

[0008] The above-mentioned Lactobacillus crispatus NCU-31 has inhibitory effects on various common pathogenic bacteria in the human body and has strong antioxidant effects. In addition, using the postbiotics of the above-mentioned Lactobacillus crispatus NCU-31 in the treatment of vulvar lichen sclerosus can significantly improve the treatment effect.

[0009] In a second aspect, the present invention provides the use of the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 in the preparation of a drug for treating vulvar lichen sclerosus.

[0010] In a third aspect, the present invention provides a drug for treating vulvar lichen sclerosus, which comprises the postbiotics of the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31.

[0011] In some alternative embodiments, the dosage form of the above-mentioned drug for treating vulvar lichen sclerosus is a liquid preparation.

[0012] In some alternative embodiments, the preparation method of the postbiotics of the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 comprises the following steps: Inoculate the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 into a liquid medium to cultivate a bacterial liquid; Centrifuge the above-mentioned bacterial liquid and collect the supernatant, and filter it with a sterile filter membrane. The obtained filtrate is the postbiotics of Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31.

[0013] In some alternative embodiments, the above-mentioned liquid medium is MRS liquid medium.

[0014] In some alternative embodiments, the temperature condition for the above-mentioned cultivation is 37 °C.

[0015] In some alternative embodiments, the temperature condition for the above-mentioned centrifugation is 4 °C and the rotation speed is 8000 rpm.

[0016] In some alternative embodiments, the above-mentioned filter membrane is a 0.22 μm microporous filter membrane. The 0.22 μm microporous filter membrane can remove bacteria and other impurities to obtain cell-free postbiotics.

[0017] In a fourth aspect, the present invention provides a drug combination for treating vulvar lichen sclerosus, which comprises the postbiotics of the above-mentioned Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 and clobetasol propionate.

[0018] When the postbiotics of the above-mentioned Lactobacillus crispatus NCU-31 are used in combination with clobetasol propionate, it can significantly promote the effect of clobetasol propionate in the treatment of vulvar lichen sclerosus.

[0019] Due to the above technical solution, the embodiments of the present invention have the following beneficial effects: A strain of Lactobacillus crispatus NCU-31 is provided, which has an inhibitory effect on a variety of common pathogenic bacteria in the human body and has a strong antioxidant effect; the postbiotics of Lactobacillus crispatus NCU-31 can be used to treat vulvar lichen sclerosus, with good treatment effect, safety and no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shows the result of Gram staining of Lactobacillus crispatus NCU-31 in an embodiment of the present invention.

[0021] Figure 2 Shows the neighbor-joining phylogenetic tree of Lactobacillus crispatus NCU-31 in an embodiment of the present invention.

[0022] Figure 3 Shows the growth characteristic curve of Lactobacillus crispatus NCU-31 in an embodiment of the present invention.

[0023] Figure 4 Shows the inhibitory effect of the postbiotics of Lactobacillus crispatus NCU-31 on the growth of common pathogenic bacteria in an embodiment of the present invention.

[0024] Figure 5 Shows the antioxidant experiment result of Lactobacillus crispatus NCU-31 in an embodiment of the present invention.

[0025] Figure 6 Shows the Cattaneo clinical symptom and sign score result in an embodiment of the present invention.

[0026] Figure 7 Shows the Chao1 index, Shannon index, Simpson index, Pielou_e index and Faith_pd index of the patients in the VG group and VGP group in an embodiment of the present invention.

[0027] Figure 8 Is a Venn diagram of the vulvar skin microbial species of the patients in the VG group and VGP group in an embodiment of the present invention.

[0028] Figure 9 Shows the results of principal coordinate analysis based on weighted UniFrac distance and non-metric multi-dimensional scaling analysis of the vulvar skin microbial community of the patients in the VG group and VGP group in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The following will clearly and completely describe the concept and technical effects generated by the present invention in combination with the embodiments and drawings, so as to fully elaborate the purpose, solution and effects of the present invention.

[0030] Lactobacillus crispatus ( Lactobacillus crispatus ) Isolation and identification of NCU-31: The vaginal secretions of healthy female volunteers and patients with vulvar lichen sclerosus from Jiangxi Provincial Maternal and Child Health Hospital were cultured and identified, and a total of 183 bacterial strains were obtained, of which Lactobacillus ( Lactobacillus spp.) bacteria, and one of the Lactobacillus species was Lactobacillus crispatus ( Lactobacillus crispatus Table 1 shows the results of bacterial screening of vaginal secretions of the two groups, indicating that the bacteria in vaginal secretions of healthy female volunteers are mainly Lactobacillus.

[0031] Table 1 Results of bacterial screening of vaginal secretions from healthy female volunteers and patients with vulvar lichen sclerosus The morphology of bacteria was observed by plate spreading and Gram staining. Figure 1 Shown are the results of Gram staining of Lactobacillus crispatus NCU-31, which clearly show that Lactobacillus crispatus NCU-31 has a long and curved rod-like morphology.

[0032] Figure 2 The Neighbor-Joining phylogenetic tree of Lactobacillus crispatus NCU-31 is shown.

[0033] Lactobacillus crispatus ( Lactobacillus crispatus )Antibacterial experiment of NCU-31: Take 1 mL containing 5×10 9 CFU of Lactobacillus crispatus NCU-31 was centrifuged at 8000rpm for 4 min, and the bottom sediment was discarded and the supernatant was collected; the supernatant was filtered through a 0.22 µm sterile filter membrane to remove the bacteria and other impurities, thereby collecting the cell-free supernatant of Lactobacillus crispatus NCU-31, which is the postbiotic. Take 100 μL containing 1×10 9 CFU of pathogenic bacteria (Staphylococcus aureus Cowan 1, Escherichia coli O157, Salmonella typhimurium ATCC 13311) were spread on a plate of LB solid culture medium, placed on an Oxford cup, 200 μL of Lactobacillus crispatus NCU-31 postbiotics were pipetted into the Oxford cup, and cultured in an incubator at 37°C. The size of the inhibition zone was observed every two hours, and the diameter was measured after 8 hours.

[0034] Figure 3 The growth characteristic curve of Lactobacillus crispatus NCU-31 is shown, indicating that the strain has good growth ability under standard culture conditions, can successfully complete the logarithmic phase proliferation and enter the stable phase, indicating that it has strong environmental adaptability and stable growth characteristics.

[0035] Figure 4 It shows the inhibitory effect of postbiotics of Lactobacillus crispatus NCU-31 on the growth of common pathogenic bacteria, indicating that the postbiotics of Lactobacillus crispatus NCU-31 have inhibitory effects on Escherichia coli O157, Salmonella typhimurium ATCC 13311, and Staphylococcus aureus Cowan I.

[0036] Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 antioxidant experiment: (1) Determination of DPPH radical scavenging ability In the experimental group, 2 mL of postbiotics of Lactobacillus crispatus NCU-31 was taken and 2 mL of 0.2 mmol / L DPPH methanol solution was added, and the reaction was carried out for 30 minutes at room temperature in the dark. After the reaction was completed, the absorbance (OD 517 ) of the solution at a wavelength of 517 nm was measured using a spectrophotometer. In the blank group, an equal amount of MRS liquid medium was used instead of the postbiotics of Lactobacillus crispatus NCU-31, and in the control group, an equal amount of deionized water was used instead of the DPPH methanol solution, and the remaining steps were the same as those in the experimental group. The formula for calculating the DPPH radical scavenging rate is: DPPH radical scavenging rate = [(A0 - (A1 - A2)) / A0] × 100% Among them, A0 represents the absorbance of the blank group (2 mL of MRS liquid medium + 2 mL of DPPH methanol solution), A1 represents the absorbance of the experimental group (2 mL of postbiotics of Lactobacillus crispatus NCU-31 + 2 mL of DPPH methanol solution), and A2 represents the absorbance of the control group (2 mL of postbiotics of Lactobacillus crispatus NCU-31 + 2 mL of deionized water).

[0037] (2) Hydroxyl radical scavenging ability In the experimental group, 1 mL of 2 mmol / L ferrous sulfate solution, 1 mL of 6 mmol / L hydrogen peroxide solution, and 1 mL of postbiotics of Lactobacillus crispatus NCU-31 were successively added to a test tube, and it was left standing for 10 min, then 2 mL of 6 mmol / L salicylic acid solution was added. At room temperature, it was left standing for 30 min, and the absorbance (OD 510 ) at 510 nm was measured. In the blank group, an equal amount of MRS liquid medium was used instead of the postbiotics of Lactobacillus crispatus NCU-31, and the remaining steps were the same as those in the experimental group. The formula for calculating the hydroxyl radical scavenging rate is: Hydroxyl radical scavenging rate = [(A0 - A1) / A0] × 100% Among them, A0 represents the absorbance of the blank group (1 mL of FeSO 4 solution + 1 mL of H 2 O 2 solution + 1 mL of medium + 2 mL of salicylic acid solution), and A1 represents the absorbance of the experimental group (FeSO 41 mL of solution + H 2 O 2 Absorbance of (1 mL of solution + 1 mL of postbiotics of Lactobacillus crispatus NCU-31 + 2 mL of salicylic acid solution).

[0038] (3) Superoxide radical scavenging ability Add 2 mL of 150 mmol / L Tris-HCl solution with pH = 8.0 and 1 mL of 1.2 mmol / L pyrogallol solution successively to 0.5 mL of postbiotics of Lactobacillus crispatus NCU-31, shake well quickly, and measure the absorbance at 330 nm (OD 330 ). Measure the absorbance every 30 s until 150 s. The formula for calculating the superoxide radical scavenging rate is: Superoxide radical scavenging rate = (A0 - A1) / A0 × 100% Among them, A0 represents the self-oxidation rate obtained by taking the slope of the fitted straight line of the absorbance value of pyrogallol's self-oxidation every 30 s, and A1 represents the oxidation rate obtained by taking the slope of the fitted straight line of the absorbance value of the mixed reaction solution of pyrogallol and postbiotics of Lactobacillus crispatus NCU-31 every 30 s.

[0039] (4) Chelating ability to ferrous ions In the experimental group, take 0.5 mL of the bacterial solution of Lactobacillus crispatus NCU-31, add 0.1 mL of 0.4% ferrous sulfate solution, mix, then add 0.1 mL of 1% vitamin C solution and 1 mL of 0.2 mol / L NaOH solution, and react at room temperature for 20 minutes; then add 1 mL of 10% trichloroacetic acid solution, centrifuge at 4°C and 6000 rpm for 10 minutes to remove proteins; take 0.4 mL of the centrifuged supernatant, add 4 mL of 0.1% o-phenanthroline solution, and react at room temperature for 10 minutes; use a spectrophotometer to measure the absorbance of the solution at a wavelength of 546 nm (OD 546 ). In the blank group, use an equal amount of MRS liquid medium instead of the bacterial solution of Lactobacillus crispatus NCU-31, and in the control group, use an equal amount of deionized water instead of ferrous sulfate solution, and the remaining steps are the same as those in the experimental group. The formula for calculating the chelating ability of ferrous ions is: Fe 2+ Chelating ability = [A0 - (A1 - A2)] / A0 × 100% Among them, A0 represents the absorbance of the blank group (0.5 mL of MRS liquid medium + 0.1 mL of 0.4% ferrous sulfate solution), A1 represents the absorbance of the experimental group (0.5 mL of the bacterial solution of Lactobacillus crispatus NCU-31 + 0.1 mL of 0.4% ferrous sulfate solution), and A2 represents the absorbance of the control group (0.5 mL of the bacterial solution of Lactobacillus crispatus NCU-31 + 0.1 mL of deionized water).

[0040] (5) Determination of total reducing power Take 1 mL of Lactobacillus crispatus NCU-31 bacterial solution, add 1 mL of PBS with pH = 6.6 and 1 mL of 1% K 3 [Fe(CN) 6 solution. After mixing, react at 50 °C for 2 minutes; add 1 mL of 10% trichloroacetic acid solution, mix well, take 1 mL, add 4 mL of deionized water and 0.4 mL of 0.1% FeCl 3 solution, and let it stand for 10 minutes. Using MRS liquid medium as the blank control, use a spectrophotometer to measure the absorbance (OD 700 ) of the solution at a wavelength of 700 nm. The formula for calculating the total reducing power is: Total reducing power = A1 - A0 where A0 represents the absorbance of the control group (1 mL of MRS liquid medium), and A1 represents the absorbance of the experimental group (1 mL of Lactobacillus crispatus NCU-31 bacterial solution). The strength of the reducing power is proportional to the absorbance of the reaction solution. The greater the absorbance, the stronger the reducing ability of the test substance.

[0041] Figure 5 The antioxidant experiment results are shown, indicating that the postbiotics of Lactobacillus crispatus NCU-31 can scavenge DPPH radicals, superoxide radicals, hydroxyl radicals, and have ferrous ion chelating ability, showing strong antioxidant ability.

[0042] Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 postbiotic treatment experiment: Inoculate the activated Lactobacillus crispatus NCU-31 into 30 mL of MRS liquid medium, culture it in a constant temperature shaker at 37 °C and 200 r / min for 24 h. Centrifuge the bacterial solution in a refrigerated high-speed centrifuge at 4 °C and 8000 rpm for 10 min, discard the bottom precipitate, collect the supernatant, take 10 - 12 mL of the supernatant and filter it through a 0.22 μm sterile filter membrane to remove bacteria and other impurities, and collect the cell-free supernatant (CFS) of Lactobacillus crispatus NCU-31, which is the postbiotic.

[0043] The clinical data of 100 patients with vulvar sclerosing lichen diagnosed by vulvar biopsy admitted to Jiangxi Maternal and Child Health Hospital from September 1, 2024 to December 31, 2024 were used. According to the random grouping criteria, they were divided into the group of clobetasol propionate combined with postbiotics of Lactobacillus crispatus NCU-31 (VGP) and the group of clobetasol propionate combined with placebo (VG). There was no significant difference in the baseline data between the two groups, indicating comparability and balance. Inclusion criteria: aged 20 - 70 years; without severe heart, lung, kidney, liver dysfunction and metabolic diseases; diagnosed as vulvar sclerosing lichen by vulvar biopsy. Exclusion criteria: pregnant patients; those with organic lesions in important organs; those with combined vulvar or cervical malignant lesions. Before agreeing to participate, each prospective participant was given a participant information sheet and the basic principles of the study were explained to them. All participants provided written informed consent.

[0044] For the group of clobetasol propionate combined with placebo, clobetasol propionate (10mg, Guangdong China Resources Shunfeng Pharmaceutical Co., Ltd.) was used according to the following regimen: once a day for 4 weeks during the induction remission phase, then once every other day for 4 weeks, and then twice a week for 4 weeks, for a total of 3 months. The dosage was referred to the fingertip unit (FTU). 1 fingertip unit refers to the amount of ointment extruded from a 5mm opening ointment tube covering from the fingertip of the index finger to the first interphalangeal joint (about 0.5g).

[0045] For the group of clobetasol propionate combined with postbiotics of Lactobacillus crispatus NCU-31, in addition to using clobetasol propionate according to the regimen of the group using only clobetasol propionate, CFS was used in the following way: clean and dry the affected area, and gently apply 100μL of CFS to the entire lesion area.

[0046] Collect the Cattaneo clinical symptom and sign scores of the two groups of patients one month after treatment. The scoring criteria are shown in Table 2.

[0047] Table 2 VLS Clinical Symptom and Sign Scoring Table Figure 6 The Cattaneo clinical symptom and sign scoring results are shown, indicating that the itching degree of the patients in the VGP group decreased, the skin elasticity improved, the skin color became more normal, and the lesion range decreased. That is to say, CFS improved the therapeutic effect of clobetasol propionate.

[0048] Collect the vulvar skin swabs of the two groups of patients one month after treatment, and use high-throughput sequencing method to detect the differences in vulvar skin flora before and after treatment in the two groups of patients. The results are as Figures 7 - 9As shown, the Chao1 index, Shannon index, Simpson index, Pielou_e index, and Faith_pd index of the VGP group patients all decreased, the types of vulvar skin microorganisms decreased, and the beneficial bacteria increased. That is to say, CFS has the ability to restore the homeostasis of the vulvar skin flora.

[0049] As described above, these are only the preferred embodiments of the present invention. The present invention is not limited to the above-mentioned implementation manners. As long as it achieves the technical effects of the present invention by the same means, it should fall within the protection scope of the present invention. Within the protection scope of the present invention, various different modifications and changes can be made to its technical solutions and / or implementation manners.

Claims

1. A strain of Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31, characterized in that, The Lactobacillus crispatus ( Lactobacillus crispatus ) NCU-31 has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is August 29, 2024, and the deposit number is CGMCC NO. 31777.

2. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) NCU-31, characterized in that, The Lactobacillus crispatus ( Lactobacillus crispatus ) The 16sRNA sequence of NCU-31 is shown as SEQ ID No.

1.

3. The Lactobacillus crispatus according to claim 1 ( Lactobacillus crispatus ) Use of NCU-31 in the preparation of a medicament for treating vulvar lichen sclerosus.

4. A drug for treating vulvar lichen sclerosus, characterized in that: comprising the Lactobacillus crispatus of claim 1 ( Lactobacillus crispatus ) epizoites of NCU-31.

5. The drug for treating vulvar lichen sclerosus according to claim 4, characterized in that: The Lactobacillus crispatus ( Lactobacillus crispatus ) The preparation method of NCU-31 postbiotics comprises the following steps: The Lactobacillus crispatus ( Lactobacillus crispatus )NCU-31 was inoculated into liquid culture medium to culture bacterial liquid; The bacterial solution was centrifuged and the supernatant was collected and filtered with a sterile filter membrane. The filtrate obtained was Lactobacillus crispatus ( Lactobacillus crispatus ) epizoites of NCU-31.

6. The drug for treating vulvar lichen sclerosus according to claim 5, characterized in that: The liquid culture medium is MRS liquid culture medium.

7. The drug for treating vulvar lichen sclerosus according to claim 5, characterized in that: The culture temperature condition is 37°C.

8. The drug for treating vulvar lichen sclerosus according to claim 5, characterized in that: The centrifugal temperature was 4°C and the rotation speed was 8000 rpm.

9. The drug for treating vulvar lichen sclerosus according to claim 5, characterized in that: The filter membrane is a 0.22 μm microporous filter membrane.

10. A drug combination for treating vulvar lichen sclerosus, characterized in that: comprising the Lactobacillus crispatus of claim 1 ( Lactobacillus crispatus ) Postbiotics and clobetasol propionate of NCU-31.

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