Application of Weissella fusiformis in preventing and treating inflammatory bowel disease

Abstract

The invention relates to the technical field of microorganisms, in particular to application of Weissella fusiformis in preventing and treating inflammatory bowel diseases. The fusion Weissella sp. Is the fusion Weissella sp. Wc1982. The application comprises one or more of resisting intestinal mucosa injury and reducing abnormal fecal characters or intestinal bleeding caused by the inflammatory bowel disease. The research finds that the Weissella Wc1982 has a remarkable function in preventing and treating the inflammatory bowel disease, can effectively relieve various symptoms of the inflammatory bowel disease, has relatively high safety, provides a new strategy for preventing and treating the inflammatory bowel disease, and has a potential clinical application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an application of Weissella fusion in preventing and treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a type of chronic, recurrent intestinal inflammatory disease. The occurrence and development of IBD is related to many factors such as genetics, abnormal immune response, intestinal barrier disorders, and gastrointestinal microbiome disorders. Currently, it mainly relies on drug therapy, surgical treatment, and lifestyle adjustments for intervention. Drug therapy mainly includes aminosalicylic acid drugs, corticosteroids, immunosuppressants, and biological agents. However, these drugs have certain side effects.

[0003] As a kind of living microorganism, probiotics have become one of the optional strategies for treating IBD by regulating the body's immune response, increasing the intestinal mucosal barrier, and improving intestinal flora disorders. Then "probiotics" is a description of the functions of these microorganisms. Probiotics actually include many different genera and species of microorganisms. However, there is no example of using Weissella fusion to treat inflammatory bowel disease. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an application of fused Weissella in preventing and treating inflammatory bowel disease.

[0005] In a first aspect, the present invention provides a fused Weissella, or a bacterial agent prepared therefrom, or a composition comprising the same, for use in treating inflammatory bowel disease; the fused Weissella is fused Weissella Wc1982.

[0006] The fusion Weissella Wc1982 described in the present invention is disclosed in Chinese Patent 202311096261.3.

[0007] The present invention further provides a use of a fused Weissella, or a bacterial agent prepared therefrom, or a composition comprising the same in the preparation of a medicament for treating inflammatory bowel disease; the fused Weissella is a fused Weissella Wc1982.

[0008] Furthermore, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0009] Furthermore, the application includes: increasing colon length, resisting intestinal mucosal damage, reducing one or more of abnormal stool characteristics or intestinal bleeding caused by inflammatory bowel disease.

[0010] Furthermore, the application includes: reducing one or more of epithelial cell shedding, inflammatory cell infiltration or crypt structure destruction caused by inflammatory bowel disease.

[0011] The present invention further provides a fused Weissella, or a bacterial agent prepared therefrom, or a composition comprising the same for use in producing one or more of alkaline phosphatase, leucine aromatic amine enzyme, acid phosphatase or naphthol-AS-BI-phosphohydrolase; the fused Weissella is fused Weissella Wc1982.

[0012] Furthermore, the number of viable bacteria of Weissella Wc1982 in the bacterial agent is 1×10 7~10 CFU / mL.

[0013] In a second aspect, the present invention provides a drug for preventing and treating inflammatory bowel disease, comprising: fusion Weissella Wc1982.

[0014] In a third aspect, the present invention provides a method for preparing a drug for preventing and treating inflammatory bowel disease, comprising: preparing the drug using Weissella fusion Wc1982.

[0015] Furthermore, the medicine is in the form of capsules, lyophilized powder or bacterial liquid preparation.

[0016] The present invention has the following beneficial effects: The present invention provides a fusion of Weissella ( Weissella confusa ) The application of Wc1982 in relieving intestinal inflammation and preventing and treating inflammatory bowel disease is highly safe for animals. It can be used as a probiotic preparation to relieve inflammatory bowel disease, reduce intestinal mucosal damage and colon inflammation, and provide a new strategy for the prevention and treatment of inflammatory bowel disease. It has potential clinical application prospects.

[0017] The colitis model of C57BL / 6J mice was verified by using fusion bacteria Weissella ( Weissella confusa ) The Wc1982 bacterial solution was pre-gavulated for 7 days, and the strain had no obvious toxic side effects on the experimental animals; after 7 days of formal DSS intervention, it was found that compared with the mice in the model group, the Wc1982 intervention group had alleviated symptoms such as diarrhea and bloody stools, a significantly decreased disease activity index, and a reduced degree of colon shortening. The results of colon tissue pathology showed that the destruction of the intestinal mucosal structure, goblet cell loss, and inflammatory cell infiltration were significantly alleviated, proving that the strain can reduce intestinal mucosal damage and inflammatory response in mice with colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a graph showing the hemolytic activity and gelatinase activity of the fused Weissella Wc1982 provided in Example 1 of the present invention; wherein A represents the hemolytic activity and B represents the gelatinase activity.

[0020] Figure 2 This is a comparative diagram of the effect of Weissella Wc1982 fusion provided in Example 2 of the present invention on the disease activity index (DAI) of DSS-induced colitis model mice; in the figure, control represents the normal control group, DSS represents the model group, and Wc1982 represents the Weissella Wc1982 fusion intervention group. The results are shown as x±SD, N=6; *: P<0.05; **: P<0.01; ****: P<0.0001, the same below.

[0021] Figure 3 This is a comparative graph of the effect of the fused Weissella Wc1982 provided in Example 2 of the present invention on the colon length of DSS-induced colitis model mice; wherein A is a representative graph of colon length, and B is a comparative graph of colon length.

[0022] Figure 4 This is a HE staining image of the pathological changes in colon tissue of DSS-induced colitis model mice induced by the fusion Weissella Wc1982 provided in Example 2 of the present invention; the figure shows a microscopic image of H&E stained pathological sections of colon tissue (scale: 200 μm; 50 μm).

[0023] Figure 5 This is a comparison chart of the colon tissue pathology score of DSS-induced colitis model mice by the fusion Weissella Wc1982 provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0026] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0027] Example 1 Characteristic Analysis of Fusion Weissella Wc1982 1. Fusion of DPPH free radical scavenging ability of Weissella Wc1982 The present invention treats the bacterial strain in the logarithmic growth phase at 10 7 After inoculation of CFU / mL of bacteria in MRS culture medium for 24 h, centrifuge at 4°C, 4000 r / min for 10 min, filter through a 0.22 μM sterile filter membrane to obtain cell-free supernatant (CFS), and prepare 0.4 mmol / L DPPH solution with anhydrous ethanol. Add 2 mL CFS and 2 mL DPPH solution to the sample group (As); add 2 mL DPPH solution and 2 mL MRS liquid culture medium to the control group (Ac); add 2 mL CFS and 2 mL anhydrous ethanol to the blank group (Ab). Incubate at 37°C in the dark for 0.5 h, and measure the absorbance at 517 nm. Calculate DPPH clearance rate by formula. Lactobacillus rhamnosus GG ATCC 53103 served as a positive control.

[0028] The results showed that the DPPH scavenging rate of Wc1982 was 79.10±1.21%, which showed a certain antioxidant capacity. The DPPH scavenging rate of the positive control bacteria was 86.35±3.38%.

[0029] 2. Hemolytic activity and gelatinase activity of Weissella Wc1982 Hemolytic activity test: The strain in logarithmic growth phase was mixed with PBS to make 10 8 CFU / mL of bacterial suspension was added, and 10 μL was dripped on the surface of MRS + 5% (w / v) fresh defibrinated sheep blood agar medium, cultured at 37 °C for 24 h, and the hemolysis phenomenon was observed to determine whether α hemolysis, β hemolysis or γ hemolysis occurred. Staphylococcus aureus ATCC 25923 served as a positive control.

[0030] Gelatinase activity detection: 10 μL of the above bacterial suspension (10 8CFU / mL), and after culturing at 37 °C for 72 h, pour saturated ammonium sulfate solution on the surface of the culture medium. If a ring-shaped transparent zone appears around the colony, it is gelatinase positive; if there is no ring-shaped transparent zone, it is gelatinase negative. S. aureus ATCC 25923 served as a positive control.

[0031] The results of the hemolytic activity test were as follows Figure 1 As shown in A, S. aureus Clearly defined, completely transparent hemolytic rings (β-hemolytic rings) can be observed around the ATCC 25923 colonies, indicating hemolytic activity, while no hemolysis is observed around the Wc1982 colonies, indicating γ-hemolysis, i.e., no hemolytic activity. Figure 1 As shown in B, S. aureus An annular transparent halo appeared around the ATCC 25923 colony, indicating gelatinase activity, while no annular transparent zone was seen around the Wc1982 colony, indicating gelatinase negative.

[0032] 3. API ZYM zymogram of Weissella Wc1982 The present invention is operated according to the instructions of the API ZYM test kit produced by BioMérieux, France, to evaluate the activity of 19 enzymes including β-glucuronidase. The results are graded from 0 to 5 by comparing the color intensity, 0 being a negative reaction, and if the result exceeds 3, it is judged to be positive.

[0033] The results are shown in Table 1. Wc1982 had no β-glucuronidase activity, but could produce alkaline phosphatase, leucine arylaminase, acid phosphatase and naphthol-AS-BI-phosphohydrolase.

[0034] Table 1 Enzyme activity of Weissella fusion Wc1982

[0035] Note: +, positive; -, negative; w, weak positive Example 2 Fusion of Weissella Wc1982 improves DSS-induced colitis in mice The DSS colitis model was constructed using C57BL / 6J mice, and Wc1982 suspension was administered intragastrically (200 μL / mouse, with a bacterial count of approximately 2×10 8 The specific experimental design is as follows: Six-week-old experimental mice were adapted to an SPF environment at 25 °C and a 12 h light / dark cycle for 3 days and fed with common rodent chow and sterilized water. They were randomly divided into three groups (N=6), namely the control group (Control group), the model group (DSS group) and the Wc1982 intervention group (Wc1982 group). They were pre-gavaged for 7 days (-7 to -1 d). The DSS group and the Wc1982 group were gavaged with 200 μL PBS and Wc1982 suspension, respectively; 3% (w / v) DSS was added to the drinking water of the two groups of mice, and gavage was continued for 7 days (0 to 6 d). The body weight, fecal characteristics and occult blood status of the mice were recorded daily, and the disease activity index (DAI) was calculated according to the scoring criteria shown in Table 2.

[0036] Table 2 Disease Activity Index Scoring Criteria

[0037] DAI = score obtained by weight loss + score obtained by stool characteristics + score obtained by occult blood status.

[0038] After the experiment, the length of the mouse colon was measured, and the distal colon was taken and immersed in 4% paraformaldehyde for subsequent histopathological analysis. The pathological scoring criteria are shown in Table 3.

[0039] Table 3 Colon histopathological scoring criteria

[0040] Note: The total score is 0-4; the range of inflammation is the basic criterion for grading, and only one of the other criteria needs to be met Table 4 and Figure 2 The results showed that from the second day, the DAI score of the Control group was significantly lower than that of the DSS group (P<0.05), which lasted until the end of the experiment; the DAI score of the Wc1982 group on the 4th and 6th days was significantly lower than that of the DSS group (P<0.05).

[0041] Table 4 DAI score results of experimental mice

[0042] Note: Compared with the model group, *, P<0.05; **, P<0.01; ****, P<0.0001.

[0043] Figure 3The effect of Weissella fusion Wc1982 on the colon length of mice with DSS-induced colitis model was shown. The colon length of mice in the DSS group was 4.83±0.59 cm, which was significantly shorter than the colon length of mice in the Control group (6.82±0.54 cm) (P<0.0001), and the colon length of the Wc1982 intervention group was 5.68±0.46 cm, which was significantly longer than the colon length of mice in the DSS group (4.83±0.59 cm) (P<0.05).

[0044] Figure 4 The results show the effect of Weissella fusion Wc1982 on the pathological changes of colon tissue in mice with DSS-induced colitis model. The pathological changes of colon tissue in mice in the DSS group were widespread ulcers in the intestinal tissue, disappearance of mucosal epithelium and lamina propria structure, a large amount of connective tissue hyperplasia, moderate to severe reduction in the number of goblet cells, accompanied by more lymphocytes, granulocytes and other inflammatory cell infiltration; a small amount of connective tissue hyperplasia was also seen in the submucosal layer, and large-scale edema was seen, the connective tissue was loosely arranged, accompanied by more lymphocytes, granulocytes and other inflammatory cell infiltration. In the Wc1982 group, epithelial cell shedding was reduced, inflammatory cell infiltration was reduced, and the degree of crypt structure destruction was milder.

[0045] Table 5 and Figure 5 The effect of Weissella fusion Wc1982 on colon histopathological scores of mice with DSS-induced colitis model was shown. The frequency of histopathological scores of 3-4 points in the DSS group was higher than that in the Control group (P<0.0001, Fisher's exact test) and the Wc1982 group (P<0.01, Fisher's exact test).

[0046] Table 5 Results of colon pathology scores of experimental mice

[0047] The above results fully demonstrate that the fused Weissella Wc1982 has the effect of improving DSS-induced colitis. Mice treated with the fused Weissella Wc1982 effectively reduced the disease activity index score, increased colon length, improved colon tissue lesions in colitis model mice, and reduced the colon pathology score of colitis model mice. Therefore, the fused Weissella Wc1982 can be used to prevent and treat colitis or to prepare drugs for preventing and treating colitis.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A use of a fused Weissella, or a bacterial agent prepared therefrom, or a composition comprising the same in the treatment of inflammatory bowel disease, characterized in that: The fused Weissella is fused Weissella Wc1982.

2. A use of a fused Weissella, or a bacterial agent prepared therefrom, or a composition comprising the same in the preparation of a medicament for treating inflammatory bowel disease, characterized in that: The fused Weissella is fused Weissella Wc1982.

3. The use according to claim 1 or 2, characterized in that: The inflammatory bowel disease is ulcerative colitis or Crohn's disease.

4. The use according to any one of claims 1 to 3, characterized in that: The application includes: increasing colon length, resisting intestinal mucosal damage, reducing one or more of abnormal stool characteristics or intestinal bleeding caused by inflammatory bowel disease.

5. The use according to any one of claims 1 to 3, characterized in that: The application includes: reducing one or more of epithelial cell shedding, inflammatory cell infiltration or crypt structure destruction caused by inflammatory bowel disease.

6. A use of a fused Weissella, or a bacterial agent prepared therefrom, or a composition comprising the same in the production of one or more of alkaline phosphatase, leucine arylamine enzyme, acid phosphatase or naphthol-AS-BI-phosphohydrolase, characterized in that: The fused Weissella is fused Weissella Wc1982.

7. The use according to any one of claims 1 to 6, characterized in that: The number of viable bacteria of Weissella fusion Wc1982 in the bacterial agent is 1×10 7~10 CFU / mL.

8. A drug for preventing and treating inflammatory bowel disease, characterized in that: include: Fusion Weissella Wc1982.

9. A method for preparing a drug for preventing and treating inflammatory bowel disease, characterized in that: include: The drug was prepared using fusion Weissella Wc1982.

Images