Application of pseudosciaena hirsuta in preparation of medicine for preventing, treating and / or improving ulcerative colitis

Abstract

The invention provides application of Pseudomonas capilliformis (Pseudomonas capilliformis) to preparation of a medicine for preventing, treating and / or improving ulcerative colitis, and belongs to the technical field of probiotic preparations. The invention further provides a preparation method of the Pseudomonas capilliformis (Pseudomonas capilliformis) and application of the Pseudomonas capilliformis (Pseudomonas capilliformis (Pseudomonas capilliformis). In the invention, the pseudosciaena hirsuta is a beneficial bacterium screened through difference analysis of excrement samples in a clinical queue (including excrement samples of healthy control, active-stage ulcerative colitis patients and remission-stage ulcerative colitis patients). Existing commercialized probiotics mainly comprise lactobacillus, bifidobacterium and the like and are not designed for patients with ulcerative colitis, but the pseudomonas hirsuta provided by the invention better conforms to intestinal flora characteristics of the patients with ulcerative colitis and is suitable for individualized treatment of the patients with ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic preparations, and in particular relates to the use of Pseudomonas aeruginosa in preparing medicines for preventing, treating and / or improving ulcerative colitis. Background Art

[0002] Ulcerative colitis is a chronic, idiopathic inflammatory disease of the colon that primarily affects the rectum, colon mucosa, and submucosa. Intestinal flora plays an important role in the development and progression of ulcerative colitis, and a number of experimental and clinical studies have also confirmed that probiotic-based therapies have a high application prospect. However, there are currently no reports on the use of Pseudoflavonifractor capillosus to treat ulcerative colitis. Summary of the invention

[0003] The purpose of the present invention is to provide the use of Pseudomonas aeruginosa in the preparation of a medicament for preventing, treating and / or improving ulcerative colitis, thus filling the blank of Pseudomonas aeruginosa in the treatment of ulcerative colitis.

[0004] The invention provides application of Pseudoflavonifractor capillosus in preparing medicine for preventing, treating and / or improving ulcerative colitis.

[0005] Preferably, the Pseudoflavonoids from ...

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

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

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

[0009] Preferably, the treating and / or improving ulcerative colitis comprises improving the shortening of the colon caused by ulcerative colitis and / or repairing damaged tight junction proteins.

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

[0011] The present invention also provides the use of Pseudoflavonifractor capillosus as a detection target in the preparation of a product for diagnosing or assisting in the 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] The present invention provides the use of Pseudoflavonifractor capillosus in the preparation of a drug for preventing, treating and / or improving ulcerative colitis; the Pseudoflavonifractor capillosus is a beneficial bacteria screened out through differential analysis of fecal samples of a clinical cohort (including fecal samples of healthy controls, patients with active ulcerative colitis and patients with remission ulcerative colitis). Existing commercial probiotic ingredients are mainly lactobacilli, bifidobacteria, etc., which are not designed for patients with ulcerative colitis, while the Pseudoflavonifractor capillosus provided by the present invention is more in line with the intestinal flora characteristics of patients with ulcerative colitis and is suitable for individualized treatment of patients with ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 This is a diagram showing the results of metagenomic sequencing of intestinal flora in stool samples;

[0017] Figure 2 This is a comparison of the improvement of Pseudomonas aeruginosa on colon shortening caused by DSS model;

[0018] Figure 3 This is a statistical result diagram of the improvement of Pseudomonas aeruginosa on colon shortening caused by DSS model;

[0019] Figure 4 This is the electrophoresis diagram of the improvement of Pseudomonas aeruginosa on tight junction protein damage caused by DSS model;

[0020] Figure 5 This is a statistical graph showing the improvement of Pseudomonas aeruginosa on tight junction protein damage caused by the DSS model. DETAILED DESCRIPTION

[0021] The invention provides application of Pseudoflavonifractor capillosus in preparing medicine for preventing, treating and / or improving ulcerative colitis.

[0022] In the specific implementation process of the present invention, the Pseudomonas aeruginosa is a beneficial bacteria screened out through differential analysis of fecal samples of a clinical cohort (including fecal samples of healthy controls, patients with active ulcerative colitis, and patients with remission ulcerative colitis). Existing commercial probiotic ingredients are mainly lactobacilli, bifidobacteria, etc., which are not designed for patients with ulcerative colitis. The Pseudomonas aeruginosa provided by the present invention is more in line with the intestinal flora characteristics of patients with ulcerative colitis and is suitable for individualized treatment of patients with ulcerative colitis.

[0023] In the specific implementation process of the present invention, the polytrichum pseudoflavonoids include polytrichum pseudoflavonoids DSM 23940, which is deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ), which is equivalent to polytrichum pseudoflavonoids ATCC 29799, which is deposited in the American Type Culture Collection (ATCC).

[0024] In the specific implementation process of the present invention, the Pseudoflavonoids from ... As shown in NO.1, specifically: tcgaacggagagctcatgacagaggattcgtccaatggattgggtttcttagtggcggacgggtgagtaacgcgtgaggaacctgcctcgga gtggggaataacagtccgaaaggactgctaataccgcataatgcagctgagtcgcatgacactggctgccaaagatttatcgctctgagatggcctcgcgtct gattagctagttggcggggtaacggcccaccaaggcgacgatcagtagccggactgagaggttggccggccacattgggactgagacacggcccagactccta cgggaggcagcagtggggaatattgggcaatgggcgcaagcctgacccagcaacgccgcgtgaaggatgaaggctttcgggttgtaaacttcttttatcaggg.

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

[0026] In the specific implementation process of the present invention, the effective viable bacteria count of Pseudomonas aeruginosa in the drug is 1×10 9 ~1×10 10 CFU / mL, further 5×10 9 CFU / mL.

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

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

[0029] In the specific implementation of the present 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 forming is an important component of the intestinal epithelial barrier. Ulcerative colitis can lead to the destruction of the tight junction structure. Therefore, repairing damaged tight junction proteins plays an important role in the induction treatment of ulcerative colitis.

[0030] The present invention also provides the use of Pseudoflavonifractor capillosus as a detection target in the preparation of a product for diagnosing or assisting in the diagnosis of ulcerative colitis.

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

[0032] In a specific implementation of the present 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 stage.

[0033] In the specific implementation of the present invention, the sample of the product is feces.

[0034] The present invention collects stool samples from healthy controls, patients with active ulcerative colitis, and patients with remission ulcerative colitis, performs metagenomic sequencing on the intestinal flora therein, and finds that compared with the healthy control group, the content of Pseudoflavonoids in the intestinal flora of patients with active ulcerative colitis feces is significantly reduced. In addition, the content of Pseudoflavonoids in the intestinal flora of patients with remission ulcerative colitis is significantly restored compared with that of patients in the active stage. Therefore, compared with the bifidobacterium derived from healthy infants and young children in the prior art, Pseudoflavonoids is more in line with the intestinal flora characteristics of patients with ulcerative colitis, and is more suitable for the individualized treatment of patients with ulcerative colitis, providing a new intervention product and scheme for the treatment of ulcerative colitis.

[0035] To further illustrate the present invention, the application of Pseudomonas aeruginosa provided by the present invention in the preparation of drugs for preventing, treating and / or improving ulcerative colitis is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] 1. Screening of Pseudomonas aeruginosa

[0038] Fecal samples were collected from healthy controls (HC), patients with active ulcerative colitis (Active), and patients with remission ulcerative colitis (Remission). The evaluation index for distinguishing patients with remission ulcerative colitis and active ulcerative colitis was the Pediatric Ulcerative Colitis Activity Index (PUCAI). When PUCAI < 10, it was determined to be in remission, and PUCAI ≥ 10 was determined to be in active stage. Metagenomic sequencing was performed on the intestinal flora in the above fecal samples, and the results are shown in Figure 1 The results showed that compared with the healthy control group, the content of Pseudomonas aeruginosa in the fecal intestinal flora of patients with active ulcerative colitis was significantly decreased. In addition, the content of Pseudomonas aeruginosa in the intestinal flora of patients with remission ulcerative colitis was significantly restored compared with that of patients in the active stage.

[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, dilute to 100mL with distilled water or deionized water, add an appropriate amount of cooked beef cubes, about 1 / 3 of the liquid height, mix well, autoclave at 121℃ for 30min, cool to below 50℃, add 1 vial of 0.5mg hemin and 1 vial of 5mg vitamin K1 per 100mL under aseptic operation, and then place in an anaerobic box containing a mixed gas to deoxygenate overnight; the mixed gas consists of the following gas percentages by volume: 80% N 2 、10%H 2 and 10% CO 2 .

[0041] The lyophilized powder of Pseudoflavonoids from ...

[0042] 3. Determination of the concentration of Pseudomonas aeruginosa

[0043] After culturing Pseudoflavonoids in an anaerobic environment for 48 hours, flocculent precipitates can be seen in the liquid culture medium. After blowing and mixing, take 200 μL of the mixture in a Columbia blood plate, spread it evenly with an L-shaped coating stick, and then culture it in an anaerobic environment for 24 to 72 hours. After that, round transparent colonies can be seen on the blood plate. Use a McFarland turbidimeter to count bacteria. Add 4 mL of sterile anaerobic phosphate buffer solution (PBS) to a sterile tube, adjust the McFarland turbidimeter to zero, then use a sterile cotton swab to scrape the colony in the blood plate, swing it up and down in the above-mentioned sterile tube, shake off the colony, and measure the turbidity again to get the specific McFarland concentration unit (McF). According to 0.5McF=1.5×10 8 The conversion formula of CFU / mL can be used to obtain the specific bacterial liquid concentration.

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

[0045] The concentration of Pseudoflavonoids in the bacterial solution obtained in step 3 was quantified to 5×10 9 CFU / mL, used for subsequent oral gavage of mice.

[0046] Dextran sulfate sodium (DSS) was used to construct a mouse ulcerative colitis model. For specific modeling methods, see [Wirtz S, Popp V, Kindermann M, et al. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat Protoc. 2017; 12(7): 1295-1309].

[0047] Thirty 8-week-old C57BL / 6J male mice were pre-fed for one week and then divided into three groups: PBS group (con), DSS group, and DSS+Pseudoflavonifractorcapillosus group, with 10 mice in each group. A 3% DSS solution was used for modeling, and 24 g of DSS powder was weighed and dissolved in sterile water, and the volume was adjusted to 800 mL. The mixture was fully dissolved and mixed, and the mixture was used immediately.

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

[0049] DSS+PBS group: Mice were given 200 μL / d of PBS by gavage for 17 days, and the drinking water was changed to DSS solution from the 10th day.

[0050] DSS+Pseudoflavonifractor capillosus group: Mice were given 200μL / d of Pseudoflavonifractor capillosus bacterial solution by gavage for 17 days, and the drinking water was changed to DSS solution from the 10th day. After the experiment, anatomical sampling was performed to measure the colon length of the mice in the natural state, and to compare whether the supplementation of Pseudoflavonifractor capillosus improved the shortening of the colon caused by the modeling, so as to evaluate the therapeutic effect. Figure 2 and Figure 3 It can be seen that supplementation with Pseudomonas aeruginosa improved the colon shortening caused by modeling.

[0051] After measuring the colon length, the expression of Claudin 1 protein in the colon tissue of each group of mice was detected to evaluate the effect of Pseudomonas aeruginosa on intestinal barrier function. 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 of tight junction proteins caused by modeling.

[0052] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of Pseudoflavonifractor capillosus in the preparation of a medicament for preventing, treating and / or improving ulcerative colitis.

2. The use according to claim 1, characterized in that: The Pseudoflavonoids of polytrichum include Pseudoflavonoids of polytrichum ATCC 29799.

3. The use according to claim 1, characterized in that: The ulcerative colitis includes dextran sulfate sodium-induced ulcerative colitis.

4. The use according to claim 1, characterized in that: The effective viable bacteria count of Pseudomonas aeruginosa in the drug is 1×10 9 ~1×10 10 CFU / mL.

5. The use according to claim 1, characterized in that: The dosage form of the drug includes an oral dosage form.

6. The use according to claim 1, characterized in that: The treating and / or improving ulcerative colitis includes improving the shortening of the colon caused by ulcerative colitis and / or repairing damaged tight junction proteins.

7. The use according to claim 6, characterized in that: The tight junction proteins include Claudin1 protein.

8. Use of Pseudoflavonifractor capillosus as a detection target in the preparation of products for diagnosis or auxiliary diagnosis of ulcerative colitis.

9. The use according to claim 8, characterized in that: The ulcerative colitis includes active ulcerative colitis.

10. The use according to claim 8, characterized in that: The diagnosis or auxiliary diagnosis of ulcerative colitis includes distinguishing healthy individuals from patients with active ulcerative colitis or distinguishing patients with remission ulcerative colitis from patients with active ulcerative colitis.

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