Use of pseudosporaxanthomonas hirsuta in preparation of a drug for preventing, treating and / or improving ulcerative colitis

Abstract

The application provides application of Pseudoflavonifractor capillosus in preparation of medicines for preventing, treating and / or improving ulcerative colitis, and belongs to the technical field of probiotic preparations.In the application, the Pseudoflavonifractor capillosus is a beneficial bacterium screened through differential analysis of clinical cohort fecal samples (including fecal samples of healthy controls, patients with active ulcerative colitis and patients with remission ulcerative colitis). Existing commercial probiotic ingredients are mainly lactobacillus and bifidobacterium, and are not designed for ulcerative colitis patients, while the Pseudoflavonifractor capillosus provided in the application is more in line with the intestinal flora characteristics of ulcerative colitis patients, and is suitable for individualized treatment of ulcerative colitis patients.

Description

Technical Field

[0001] This invention belongs to the field of probiotic preparation technology, and particularly relates to the application of *Pseudomonas polychaete* in the preparation of drugs for the prevention, treatment and / or improvement of ulcerative colitis. Background Technology

[0002] Ulcerative colitis is a chronic, idiopathic inflammatory bowel disease that primarily affects the rectal and colonic mucosa and submucosa. The gut microbiota plays a crucial role in the development and progression of ulcerative colitis, and numerous experimental and clinical studies have demonstrated the promising potential of probiotic-based therapies. However, there are currently no reports of using *Pseudolaricis polychaete* to treat ulcerative colitis. Summary of the Invention

[0003] The purpose of this invention is to provide the application of *Pseudomonas polychaete* in the preparation of medicaments for the prevention, treatment and / or improvement of ulcerative colitis, filling the gap in the treatment of ulcerative colitis with *Pseudomonas polychaete*.

[0004] This invention provides the use of *Pseudoflavonifractorcapillosus* in the preparation of medicaments for the prevention, treatment, and / or improvement of ulcerative colitis.

[0005] Preferably, the *Pseudomonas polychaete* includes *Pseudomonas polychaete* ATCC 29799.

[0006] Preferably, the ulcerative colitis includes ulcerative colitis induced by sodium dextran sulfate.

[0007] Preferably, the effective viable count of *Pseudomonas polychaete* in the drug is 1 × 10⁻⁶. 9 ~1×10 10 CFU / mL.

[0008] Preferably, the dosage form of the drug includes an oral dosage form.

[0009] Preferably, the treatment and / or improvement of ulcerative colitis includes improving colonic shortening caused by ulcerative colitis and / or repairing damaged tight junction proteins.

[0010] Preferably, the tight junction protein includes Claudin 1 protein.

[0011] This invention also provides the application of *Pseudoflavonifractorcapillosus* as a detection target in the preparation of products for the diagnosis or auxiliary diagnosis of ulcerative colitis.

[0012] Preferably, the ulcerative colitis includes active ulcerative colitis.

[0013] Preferably, the diagnosis or auxiliary diagnosis of ulcerative colitis includes distinguishing between healthy individuals and patients with active ulcerative colitis, or distinguishing between patients with remission ulcerative colitis and patients with active ulcerative colitis.

[0014] This invention provides the application of *Pseudoflavonifractorcapillosus* in the preparation of drugs for the prevention, treatment, and / or improvement of ulcerative colitis. The *Pseudoflavonifractorcapillosus* is a beneficial bacterium screened through differential analysis of fecal samples from clinical cohorts (including fecal samples from healthy controls, patients with active ulcerative colitis, and patients with remission ulcerative colitis). Existing commercially available probiotics mainly consist of lactobacilli and bifidobacteria, which are not specifically designed for ulcerative colitis patients. The *Pseudoflavonifractorcapillosus* provided by this invention better matches the intestinal flora characteristics of ulcerative colitis patients and is suitable for individualized treatment of ulcerative colitis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a diagram showing the results of metagenomic sequencing of gut microbiota in a fecal sample.

[0017] Figure 2 Comparative images of actual samples showing the improvement of colonic shortening induced by *Pseudomonas aeruginosa* in the DSS model;

[0018] Figure 3 A statistical graph showing the improvement of colonic shortening induced by *Pseudomonas aeruginosa* in the DSS model;

[0019] Figure 4 Electrophoresis diagram showing the improvement of tight junction protein damage induced by *Pseudomonas polychaete* in the DSS model;

[0020] Figure 5 A statistical figure showing the improvement of tight junction protein damage induced by *Pseudomonas polychaete* in the DSS model. Detailed Implementation

[0021] This invention provides the use of *Pseudoflavonifractorcapillosus* in the preparation of medicaments for the prevention, treatment, and / or improvement of ulcerative colitis.

[0022] In the specific implementation of this invention, the *Pseudomonas polychaete* is a beneficial bacterium selected through differential analysis of fecal samples from clinical cohorts (including fecal samples from healthy controls, patients with active ulcerative colitis, and patients with ulcerative colitis in remission). Existing commercially available probiotics mainly consist of lactobacilli and bifidobacteria, which are not specifically designed for ulcerative colitis patients. The *Pseudomonas polychaete* provided by this invention, however, better matches the intestinal flora characteristics of ulcerative colitis patients and is suitable for individualized treatment of ulcerative colitis.

[0023] In the specific implementation of this invention, the *Pseudomonas polychaete* includes *Pseudomonas polychaete* DSM 23940, deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), which is equivalent to *Pseudomonas polychaete* ATCC 29799, deposited at the American Type Culture Collection (ATCC).

[0024] In the specific implementation of this invention, the *Pseudomonas polychaete* DSM 23940 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number B284564. *Pseudomonas polychaete* DSM 23940 is rod-shaped or curved rod-shaped, anaerobic, and does not produce spores. The nucleic acid sequence of *Pseudomonas polychaete* DSM 23940 was determined by Hangzhou Weishu Biotechnology Co., Ltd. using the bacterial 16S rRNA gene sequencing identification method, as shown in SEQ ID. As shown in NO.1, specifically: tcgaacggagagctcatgacagaggattcgtccaatggattgggtttcttagtggcggacgggtgagtaacgcgtgaggaacctgcctcgga gtggggaataacagtccgaaaggactgctaataccgcataatgcagctgagtcgcatgacactggctgccaaagatttatcgctctgagatggcctcgcgtct gattagctagttggcggggtaacggcccaccaaggcgacgatcagtagccggactgagaggttggccggccacattgggactgagacacggcccagactccta cgggaggcagcagtggggaatattgggcaatgggcgcaagcctgacccagcaacgccgcgtgaaggatgaaggctttcgggttgtaaacttcttttatcaggg.

[0025] In the specific implementation of this invention, the ulcerative colitis includes ulcerative colitis induced by sodium dextran sulfate.

[0026] In the specific implementation of this invention, the effective viable count of *Pseudomonas polychaete* in the drug is 1 × 10⁻⁶. 9 ~1×10 10 CFU / mL, further increased to 5 × 10 9 CFU / mL.

[0027] In the specific implementation of this invention, the dosage form of the drug includes an oral dosage form; the oral dosage form includes oral liquid, capsule or granule.

[0028] In the specific implementation of this invention, the treatment and / or improvement of ulcerative colitis includes improving colonic shortening caused by ulcerative colitis and / or repairing damaged tight junction proteins.

[0029] In the specific implementation of this invention, the tight junction protein includes Claudin 1 protein. Claudin 1 protein is a tight junction protein, and the tight junction structure it participates in is an important component of the intestinal epithelial barrier. Ulcerative colitis leads to the destruction of tight junction structures; therefore, repairing damaged tight junction proteins plays an important role in the induction therapy of ulcerative colitis.

[0030] This invention also provides the application of *Pseudoflavonifractorcapillosus* as a detection target in the preparation of products for the diagnosis or auxiliary diagnosis of ulcerative colitis.

[0031] In the specific implementation of this invention, the ulcerative colitis includes active ulcerative colitis.

[0032] In the specific implementation of this invention, the diagnosis or auxiliary diagnosis of ulcerative colitis includes distinguishing between healthy individuals and patients with ulcerative colitis, or distinguishing between patients with ulcerative colitis in remission and patients with ulcerative colitis in active phase.

[0033] In the specific implementation of this invention, the sample for testing the product is feces.

[0034] This invention collects fecal samples from healthy controls, patients with active ulcerative colitis, and patients with ulcerative colitis in remission, and performs metagenomic sequencing on the gut microbiota. The results show that compared to the healthy control group, the content of *Pseudomonas aeruginosa* in the fecal gut microbiota of patients with active ulcerative colitis is significantly reduced. Furthermore, the content of *Pseudomonas aeruginosa* in the gut microbiota of patients with remission is significantly restored compared to patients in the active phase. Therefore, compared to *Bifidobacterium* derived from healthy infants in existing technologies, *Pseudomonas aeruginosa* is more consistent with the gut microbiota characteristics of ulcerative colitis patients and is more suitable for individualized treatment of ulcerative colitis, providing a new intervention product and solution for the treatment of ulcerative colitis.

[0035] To further illustrate the present invention, the application of *Pseudomonas polychaete* provided by the present invention in the preparation of medicaments for the prevention, treatment and / or improvement of ulcerative colitis is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] 1. Screening of *Pseudomonas polychaete*

[0038] Fecal samples were collected from healthy controls (HC), patients with active ulcerative colitis (Active), and patients with remission ulcerative colitis (Remission). The assessment index for distinguishing between patients with remission and active ulcerative colitis was the Pediatric Ulcerative Colitis Disease Activity Index (PUCAI). A PUCAI < 10 was defined as remission, and a PUCAI ≥ 10 was defined as active. Metagenomic sequencing was performed on the gut microbiota in the above fecal samples. The results are shown in […]. Figure 1 The results showed that, compared with the healthy control group, the content of *Pseudomonas polychaete* in the fecal intestinal flora of patients with active ulcerative colitis was significantly decreased. Furthermore, the content of *Pseudomonas polychaete* in the intestinal flora of patients with remission ulcerative colitis was significantly restored compared with those in the active phase.

[0039] 2. Cultivation and Identification

[0040] Minced meat carbohydrate broth basal medium (CMC, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.). Weigh 5.75g of the medium and dilute to 100mL with distilled or deionized water. Add an appropriate amount of minced beef granules, about 1 / 3 of the liquid height. Mix well and autoclave at 121℃ for 30min. Cool to below 50℃. Under aseptic conditions, add one 0.5mg vial of heme chloride and one 5mg vial of vitamin K1 per 100mL. Then place in an anaerobic chamber containing a mixed gas overnight for deoxygenation. The mixed gas consists of the following gases in volume percentages: 80% N2, 10% H2, and 10% CO2.

[0041] The lyophilized powder of *Pseudomonas polychaete* (ATCC 29799, DSM 23940, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.) was transferred to deoxygenated liquid culture medium, fully activated, and cultured for 48 h. Then, it was streaked onto Columbia blood agar plates in an anaerobic chamber and cultured under anaerobic conditions for another 48 h. Single colonies were picked for identification. The nucleotide sequence of the 16S rRNA gene of *Pseudomonas polychaete* DSM23940 is shown in SEQ ID NO.1.

[0042] 3. Concentration determination of *Pseudomonas polychaete*

[0043] After 48 hours of anaerobic culture of *Pseudomonas polychaete*, flocculent precipitate was observed in the liquid culture medium. After thorough mixing, 200 μL of the mixture was transferred to Columbia blood agar plates and spread evenly using an L-shaped spreader. The plates were then incubated anaerobically for 24–72 hours, after which round, transparent colonies appeared on the blood agar plates. Bacterial counting was performed using a McFarland turbidimeter. 4 mL of sterile anaerobic phosphate-buffered saline (PBS) was added to a sterile tube, and the McFarland turbidimeter was zeroed. Colonies were then scraped from the blood agar plate with a sterile swab and transferred to the aforementioned sterile tube. The colonies were shaken off, and the turbidity was measured again to obtain the specific McFarland concentration (McF). Based on the formula 0.5 McFarland = 1.5 × 10⁻⁶, the McFarland concentration was determined. 8 The conversion formula for CFU / mL can be used to obtain the specific bacterial concentration.

[0044] 4. Verify the ameliorative effect of *Pseudomonas aeruginosa* on ulcerative colitis.

[0045] The concentration of *Pseudomonas polychaete* in the bacterial culture obtained in step 3 was quantified to 5 × 10⁻⁶. 9 CFU / mL was used for subsequent gavage in mice.

[0046] A mouse model of ulcerative colitis was constructed using sodium dextran sulfate (DSS). For specific modeling methods, please refer to [Wirtz S, Popp V, Kindermann M, et al. Chemically induced mouse models of acute and chronic intestinal inflammation. NatProtoc. 2017; 12(7):1295-1309].

[0047] Thirty 8-week-old male C57BL / 6J mice were pre-fed for one week and then divided into three groups: PBS group (con), DSS group, and DSS+PBS group (n=10 per group). For model establishment, a 3% DSS solution was used. 24g of DSS powder was weighed and dissolved in sterile water, and the solution was brought to a final volume of 800mL. The solution was thoroughly dissolved and mixed, and used immediately.

[0048] PBS group: Mice were given 200 μL / d of PBS by gavage for 17 days. Starting from day 10, their drinking water was changed to sterile water.

[0049] DSS+PBS group: Mice were given 200 μL / d of PBS by gavage for 17 days. Starting from day 10, drinking water was replaced with DSS solution.

[0050] The DSS+Pseudoflavonifractor capillosus group: Mice were administered 200 μL / day of Pseudoflavonifractor capillosus bacterial solution via gavage for 17 days. From day 10 onwards, drinking water was replaced with DSS solution. After the experiment, dissection was performed to measure the colon length of the mice in their natural state. The treatment effect was evaluated by comparing whether supplementation with Pseudoflavonifractor capillosus improved the colonic shortening induced by the model. Results are as follows: Figure 2 and Figure 3 It is evident that supplementation with Pseudomonas aeruginosa improved the colonic shortening caused by the model.

[0051] After measuring colon length ， The expression levels of Claudin 1 protein in the colon tissue of mice in each group were detected to assess the effect of *Pseudomonas polychaete* on intestinal barrier function. Results are as follows: Figure 4 and Figure 5 It can be seen that after supplementing with *Pseudomonas aeruginosa*, the expression level of Claudin 1 protein was upregulated, indicating that *Pseudomonas aeruginosa* alleviated the damage to tight junction proteins caused by modeling.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of *Pseudoflavonifractor capillosus* in the preparation of drugs for the prevention, treatment and / or improvement of ulcerative colitis; The *Pseudomonas polychaete* species is *Pseudomonas polychaete* ATCC 29799. The ulcerative colitis mentioned is ulcerative colitis induced by sodium dextran sulfate.

2. The application according to claim 1, characterized in that, The effective viable count of *Pseudomonas polychaete* in the drug is 1×10⁻⁶. 9 ~1×10 10 CFU / mL.

3. The application according to claim 1, characterized in that, The dosage form of the drug includes oral dosage forms.

4. The application according to claim 1, characterized in that, The treatment and / or improvement of ulcerative colitis includes improving colonic shortening caused by ulcerative colitis and / or repairing damaged tight junction proteins.

5. The application according to claim 4, characterized in that, The tight junction protein includes Claudin 1 protein.

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