Application of deoxyribonuclease I in preparation of medicine for treating ulcerative colitis complicated with fusobacterium nucleatum infection

Abstract

The invention provides application of deoxyribonuclease I in preparation of a medicine for treating ulcerative colitis complicated with fusobacterium nucleatum infection. The invention finds that the deoxyribonuclease I can improve the conditions of weight loss and disease activity index reduction of mice, improve the conditions of colonic enlargement and colon shortening, reduce the expression levels of inflammatory factors AIM2, GSDMD and IL-1beta, improve the inflammation of colitis, improve the expression levels of barrier factors ZO-1 and occuldin, and improve the condition of barrier injury of colitis. Therefore, it is proved that the deoxyribonuclease I can be used for treating ulcerative colitis combined with fusobacterium nucleatum infection, new theoretical basis and experimental basis are provided for development of new drugs for clinically preventing and treating ulcerative colitis combined with fusobacterium nucleatum infection, and the deoxyribonuclease I has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of deoxyribonuclease I in preparing medicine for treating ulcerative colitis combined with Fusobacterium nucleatum infection. Background Art

[0002] Ulcerative colitis (UC) is a chronic, recurrent inflammatory disease with an unknown pathogenesis. Its main clinical manifestations include bloody diarrhea, abdominal cramps, and fever. Its chronic, lifelong recurring characteristics lead to a low quality of life for most UC patients and psychological problems of varying degrees. The disease is difficult to cure, with a high recurrence rate after cure. It is closely related to the incidence of colon cancer, especially because of its wide range of lesions and long course. It is recognized as a precancerous lesion of colon cancer. It is generally believed that after a course of 15 to 20 years, the risk of cancer increases by about 1% each year. Therefore, UC is listed as one of the modern intractable diseases by the World Health Organization.

[0003] The pathogenesis of UC is complex. Currently, most scholars believe that UC is a complex result of the interaction of four major factors: genetics, environment, immunity, and intestinal flora imbalance. The emergence of the "oral-intestinal axis" theory provides new insights into the pathogenesis of UC. Oral pathogens can enter the intestine through blood circulation or swallowing, breaking the inherent ecological balance of the intestine and disrupting the normal physiological function of the intestine. Fusobacterium nucleatum (Fn), as a common oral pathogen, has been proven to promote the development and metastasis of colon cancer and produce toxins to induce cell carcinogenesis. Therefore, for patients with ulcerative colitis and Fusobacterium nucleatum infection, there is an urgent need for drugs that can effectively treat ulcerative colitis and Fusobacterium nucleatum infection.

[0004] Deoxyribonuclease I (DNase I) is an endonuclease that can digest single-stranded or double-stranded DNA to produce single deoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides. Since DNase I is an enzyme that exists in the body itself, it has a high safety in clinical use and has been used clinically to treat diseases such as cystic pulmonary fibrosis. However, there are currently no reports on the use of DNase I in the treatment of ulcerative colitis combined with Fusobacterium nucleatum infection. Summary of the invention

[0005] The invention provides the use of deoxyribonuclease I in preparing a medicine for treating ulcerative colitis combined with Fusobacterium nucleatum infection, and provides a new drug treatment approach for the prevention and treatment of ulcerative colitis combined with Fusobacterium nucleatum infection.

[0006] The present invention also provides a preparation of a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection, wherein the therapeutic active ingredient includes the above-mentioned deoxyribonuclease I. The preparation has a therapeutic effect on ulcerative colitis combined with Fusobacterium nucleatum infection, expands the scope of use of deoxyribonuclease I, and has important medical significance.

[0007] The first aspect of the present invention provides the use of deoxyribonuclease I in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection.

[0008] Deoxyribonuclease I (DNase I) is an endonuclease that can digest single-stranded or double-stranded DNA to produce monodeoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides.

[0009] Fusobacterium nucleatum (Fn) is a common oral pathogen. One of the important ways in which this oral pathogen interacts with the host is through its metabolites. The extrabacterial vesicles (Fn vesicles) released by Fusobacterium nucleatum form a stable "protective shield" with the help of the lipid bilayer structure, which makes it easier for the toxic components secreted by Fusobacterium nucleatum to evade the recognition of the host defense mechanism and facilitates precise transport to distant host cells. The presence of extrabacterial vesicles (Fn vesicles) expands the scope of pathogenicity and aggravates the course of the disease.

[0010] The ulcerative colitis in the present invention is ulcerative colitis induced by dextran sulfate sodium.

[0011] The present inventors have found through extensive research that the presence of Fn vesicles can aggravate the inflammation of ulcerative colitis.

[0012] Based on a large number of previous explorations, the inventors found that the above-mentioned deoxyribonuclease I can be used as a single drug, and has a very obvious therapeutic effect on the treatment of ulcerative colitis combined with Fusobacterium nucleatum infection, and the effect is remarkable. The inventors' research shows that deoxyribonuclease I can improve the symptoms such as weight loss, reduced disease activity index, colon enlargement, and colon shortening caused by ulcerative colitis combined with Fusobacterium nucleatum infection. Therefore, deoxyribonuclease I is used to prepare a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection.

[0013] Further, the drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection is a unit preparation. The drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection containing the deoxyribonuclease I is a unit preparation, which is more conducive to use in clinical treatment. In some embodiments, the drug in the unit preparation may contain deoxyribonuclease I at a concentration of 80U / mL to 120U / mL, or the dosage may be determined according to the course of the patient's disease.

[0014] Furthermore, the drug uses deoxyribonuclease I as an active ingredient.

[0015] The drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection of the present invention has deoxyribonuclease I as an active ingredient and can alleviate ulcerative colitis combined with Fusobacterium nucleatum infection.

[0016] Furthermore, the medicine also includes excipients acceptable in pharmacy, food science or health care products science.

[0017] The present invention does not specifically limit the above-mentioned adjuvants, and suitable pharmaceutical preparations can be selected according to the selection of specific dosage forms. Examples of available adjuvants include excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, honey, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, dextrin and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium, etc.), binders (e.g., gelatin, polyvinyl pyrrolidone and polyethylene glycol), lubricants (e.g., talc, calcium stearate, magnesium stearate, spermaceti, boric acid, sodium benzoate, leucine), stabilizers (methyl parahydroxybenzoate, propyl parahydroxybenzoate, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants and spices), etc.

[0018] The present invention does not limit the specific preparation form of the drug, and the specific preparation form can be selected according to needs. Further, the preparation form of the drug can be any one of injection, oral solution, capsule, tablet or spray.

[0019] Furthermore, the deoxyribonuclease I is used to prepare a drug for improving weight loss and reducing disease activity index.

[0020] Furthermore, the deoxyribonuclease I is used to prepare a drug for improving colon enlargement and colon shortening.

[0021] Furthermore, the deoxyribonuclease I is used to prepare a drug for reducing the expression levels of inflammatory factors AIM2, GSDMD and IL-1β and improving colitis inflammation.

[0022] Furthermore, the deoxyribonuclease I is used to prepare a drug for increasing the expression levels of barrier factors ZO-1 and occludin and improving the barrier damage in colitis.

[0023] The dosage of the above-mentioned drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection varies depending on the patient's condition and other factors.

[0024] The second aspect of the present invention provides a preparation of a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection, wherein the therapeutically active ingredient comprises the deoxyribonuclease I.

[0025] In summary, the solution of the present invention has at least the following effects:

[0026] The present invention develops a new use of deoxyribonuclease I, and deoxyribonuclease I is used in the preparation of a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection. Studies have shown that deoxyribonuclease I, as an effective ingredient of a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection, can improve the weight loss of mice and the reduction of the disease activity index, improve the enlargement of the colon and the shortening of the colon, reduce the expression levels of inflammatory factors AIM2, GSDMD and IL-1β, improve colitis inflammation, increase the expression levels of barrier factors ZO-1 and occludin, and improve colitis barrier damage. The present invention confirms that deoxyribonuclease I can be used to treat ulcerative colitis combined with Fusobacterium nucleatum infection, provides a new theoretical basis and experimental basis for the development of new drugs for the clinical prevention and treatment of ulcerative colitis combined with Fusobacterium nucleatum infection, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 or the description of the prior art 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 work.

[0028] Figure 1 To identify the purity of Fusobacterium nucleatum (Fn) vesicles;

[0029] in, Figure 1 A is a transmission electron microscopy image of Fn vesicles;

[0030] B is the Western Blot result of Fn vesicles;

[0031] C is the purity diagram of Fn vesicles identified by nanoparticle tracking analysis.

[0032] Figure 2 The effect of Fn vesicles on the human normal colon epithelial cell line NCM460;

[0033] in, Figure 2 A is the Western Blot result of inflammatory factors in NCM460 cells of each group;

[0034] Figure 2B is a statistical chart of the expression of inflammatory factors in each group of NCM460 cells.

[0035] Figure 3 The effect of DNase I on the disease activity index of mice with ulcerative colitis and Fusobacterium nucleatum infection;

[0036] in, Figure 3 A is the body weight of mice in each group during 3% DSS treatment, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the group treated with Fn vesicles and DNase I, and DSS+DNase I represents the group treated with DNase I;

[0037] Figure 3 B is the disease activity index (DAI) of each group of mice during 3% DSS treatment, where DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I.

[0038] Figure 4 The effect of DNase I on the colon of mice with ulcerative colitis and infection with Fusobacterium nucleatum;

[0039] in, Figure 4 A is the colon length of mice in each group, where DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I;

[0040] B is a comparison of the significant differences in colon length among the mice in each group, *** represents p<0.001, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I.

[0041] Figure 5The figure is the hematoxylin and eosin staining (H&E staining) of the colon tissue of each group of mice, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I.

[0042] Figure 6 A is the immunohistochemical staining (IHC staining) of the colon tissue of each group of mice, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I;

[0043] Figure 6 B is a statistical graph of the expression levels of inflammatory factors in the colon tissues of each group of mice, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I.

[0044] Figure 7 A represents the expression levels of barrier factors ZO-1 and occludin in colon tissue measured by immunohistofluorescence staining (IF), wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I;

[0045] Figure 7 B is a statistical graph of the expression levels of barrier factors ZO-1 and occludin in colon tissue, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I. DETAILED DESCRIPTION

[0046] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. If the specific technology or conditions are not indicated in the embodiments, the technology or conditions described in the literature in this area or the product instructions are carried out. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0047] As used herein, the term "treat" refers to ameliorating a disease or condition (i.e., slowing down or preventing or alleviating the development of a disease or at least one of its clinical symptoms) in some embodiments. In other embodiments, "treat" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treat" refers to regulating a disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. In other embodiments, "treat" refers to preventing or delaying the onset, occurrence, or worsening of a disease or condition.

[0048] The application of the deoxyribonuclease I provided by the present invention in the preparation of a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1 Deoxyribonuclease I Inhibition of Ulcerative Colitis Combined with Fusobacterium nucleatum Infection

[0050] 1. Experimental Materials

[0051] The human normal colon epithelial cell line NCM460 was preserved in our laboratory (Key Laboratory of the Ministry of Education, School of Laboratory Medicine, Chongqing Medical University); Fusobacterium nucleatum (Fn) (ATCC 25586) was purchased from the American Standard Bacteria Bank; DMEM high-glucose complete medium was purchased from Chongqing Bopei Biotechnology Co., Ltd.; fetal bovine serum was purchased from CLARK Bioscience, USA; Western blot related reagents were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; rabbit anti-human AIM2 antibody and rabbit anti-mouse AIM2 antibody were purchased from Abcam, UK; mouse anti-human AIM2 antibody and mouse anti-mouse AIM2 antibody were purchased from SantaCruz, USA; rabbit anti-human GSDMD antibody, Cleaved caspase-1 antibody, ZO-1 antibody, and Occludin antibody were purchased from Proteintech, USA; rabbit anti-mouse GSDMD antibody was purchased from Immunoway, USA; rabbit anti-human Cleaved-IL-1β antibody and rabbit anti-mouse Cleaved-IL-1β were purchased from cell line, USA The antibodies were purchased from Biotechnology Information Corporation (Institute for Integrative Medicine, Chinese Academy of Sciences); rabbit anti-human cleaved-IL-18 and rabbit anti-mouse cleaved-IL-18 were purchased from Cell Applications Inc., USA; rabbit anti-human CD63 antibody, Alix antibody and Calnexin antibody were purchased from Wanlei Biotechnology Co., Ltd.; mouse anti-human β-actin antibody was purchased from Proteintech, USA; deoxyribonuclease I (DNase I) was purchased from Sigma, USA; dextran sulfate sodium salt (DSS) was purchased from Yishen Biotechnology Co., Ltd., China.

[0052] 2. Culture of human normal colon epithelial cell line NCM460

[0053] The human normal colon epithelial cell line NCM460 was an adherent cell. The human normal colon epithelial cell line NCM460 was inoculated into DMEM high-glucose complete medium (containing 10% fetal bovine serum and 1% double antibody) and placed at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator for 2 days and then digested with 0.25% pancreatic enzyme (Cat. No. DW0007, purchased from Shanghai Duowo Biological) and serially passaged. After 4 passages, the cultured human normal colon epithelial cell line NCM460 (cell NCM460) was obtained.

[0054] 3. Cultivation of Fusobacterium nucleatum (Fn)

[0055] Fn was cultured in 200 mL brain heart infusion (BHI) containing 0.2 mL hemin (5 mg / mL), 0.5 g potassium dihydrogen phosphate (final concentration: 2.5 g / L), 0.2 mL 0.1% vitamin K1 solution and 0.06 g L-cysteine ​​(final concentration: 0.3 g / L), and cultured at 37°C under anaerobic conditions for 48 h; then centrifuged in an ultracentrifuge at 4°C and 2500 r / min for 10 min, and the precipitate was obtained after centrifugation. The precipitate was washed twice with sterile PBS to prepare a bacterial suspension.

[0056] The turbidity of the bacterial suspension was measured using an ultraviolet spectrophotometer and its concentration was adjusted to 1 × 10 9 CFU / mL (OD600 = 1) to obtain the bacterial suspension. All subsequent experiments were treated with a multiplicity of infection (MOI) of 100:1 for NCM460 cells, where MOI represents the ratio of bacterial suspension to NCM460 cells.

[0057] 4. Extraction and purity identification of Fusobacterium nucleatum (Fn) vesicles

[0058] Fn was cultured in 200 mL brain heart infusion (BHI) containing 0.2 mL hemin (5 mg / mL), 0.5 g potassium dihydrogen phosphate (final concentration: 2.5 g / L), 0.2 mL 0.1% vitamin K1 solution and 0.06 g L-cysteine ​​(final concentration: 0.3 g / L), and cultured at 37°C under anaerobic conditions for 48 h; then centrifuged in an ultracentrifuge at 4°C and 2500 r / min for 20 min to obtain the supernatant, and the supernatant was filtered and sterilized with a 0.22 μm syringe filter (Product No.: FF372-10 pcs, Biyuntian) to finally obtain Fn conditioned medium.

[0059] Collect 40 mL of Fn-conditioned medium, and collect the extracellular vesicles of Fusobacterium nucleatum (Fn vesicles) released in the Fn-conditioned medium according to the instructions of the bacterial membrane vesicle isolation kit BacMV40-10 of Runji Bio, China, and wash with 10 mL of double distilled water (ddHO). 2 O) dilute Fn vesicles to measure the particle size and concentration of Fn vesicles; use 100 nm polystyrene particles to calibrate the ZetaView system, the temperature is maintained at 23 ° C, and the PMX 120 (Particle Metrix, Germany) and its corresponding software ZetaView 8.05.12 were used to analyze the particle size and concentration of Fn vesicles. The results are shown in Figure 1As shown in C; the microstructure of Fn vesicles was analyzed by transmission electron microscopy (TEM). The specific operation was as follows: 10 μl of Fn vesicles were dropped on the copper mesh, and the excess liquid was absorbed with filter paper after 10 minutes. Then 10 μL of 2% uranyl acetate was dropped on the copper mesh, and the excess liquid was absorbed with filter paper after 3 minutes. After drying for 5 minutes, the sample was observed with an electron microscope (Japan JEM-1200EX). The results are shown in Figure 1 As shown in A; the Fn vesicle marker protein on the Fn vesicle was detected by Western Blot, and the total protein of the human normal colon epithelial cell line NCM460 without any treatment was extracted as a control. The results are shown in Figure 1 As shown in B.

[0060] Figure 1 A is a transmission electron microscopy image of Fn vesicles; Figure 1 B is the Western Blot result of Fn vesicles; Figure 1 C is a graph showing the purity of Fn vesicles identified by nanoparticle tracking analysis (NTA).

[0061] Depend on Figure 1 As shown in A, there are Fn vesicles in the Fn conditioned medium, and the morphology of the Fn vesicles under transmission electron microscopy is a saucer-shaped structure.

[0062] Depend on Figure 1 As shown in B, compared with the control, the vesicle-specific positive proteins Alix and CD63 were expressed in the Fn vesicles, while the negative protein Calnexin was not expressed. The Western Blot results confirmed that the purity of the Fn vesicles extracted from the Fn conditioned medium was high and subsequent experiments could be carried out.

[0063] Fn vesicles were quantified by nanoparticle tracking analysis. Figure 1 C showed that the particle size distribution of Fn vesicles was between 0-200 nm, and the average particle size distribution was 100 nm.

[0064] 5. Effects of Fn vesicles on human normal colon epithelial cell line NCM460

[0065] (1) Experimental methods:

[0066] The steps for extracting cell proteins are as follows:

[0067] ①Select separation gel and concentration gel;

[0068] ② Load protein at about 180 μg / well;

[0069] ③SDS-PAGE electrophoresis (concentrating gel 90V, 30min, separating gel 120V, 1h);

[0070] ④ Transfer the membrane in the order from negative electrode to positive electrode, stack the sponge, 3 layers of filter paper, protein gel, PVDF membrane, 3 layers of filter paper, and sponge in the wet transfer clip from bottom to top; then fasten the wet transfer clip and put it into the wet transfer instrument with freshly prepared 1× wet transfer buffer, with a constant current of 210mA;

[0071] ⑤ Remove the PVDF membrane and place it in 5% BSA by mass and block it at 37°C for 2h;

[0072] ⑥ Dilute the required primary antibody with primary antibody diluent, apply the primary antibody fully on the PVDF membrane, and incubate overnight at 4°C; the antibodies (primary antibodies) used are as follows: rabbit anti-human AIM2 antibody, dilution ratio is 1:1000; rabbit anti-human GSDMD antibody, dilution ratio is 1:5000; Cleaved caspase-1 antibody, dilution ratio is 1:1000; rabbit anti-human Cleaved-IL-1β, dilution ratio is 1:1000; rabbit anti-human Cleaved-IL-18, dilution ratio is 1:1000; rabbit anti-human CD63 antibody, dilution ratio is 1:2000; Alix antibody, dilution ratio is 1:2000, Calnexin antibody, dilution ratio is 1:5000; mouse anti-human β-actin antibody, dilution ratio is 1:50000;

[0073] ⑦ On the second day, wash the membrane three times with 1×TBST on a shaker, 10 min each time;

[0074] ⑧ Dilute the secondary antibody at 1:5000 and incubate at 37°C for 1.5 h; the antibodies (secondary antibodies) used are as follows: horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (ZB-2301, Zhongshan Jinqiao), the dilution ratio is 1:5000; horseradish enzyme-labeled goat anti-mouse IgG (H+L) (affinity purification) (ZB-2305, Zhongshan Jinqiao), the dilution ratio is 1:5000;

[0075] ⑨Wash the membrane in the same way as step ⑦;

[0076] ⑩Apply ECL luminescent liquid on the PVDF membrane, develop and collect images.

[0077] (2) Experimental groups:

[0078] According to different treatment factors, the experiment was divided into control group, Fn vesicle group, and Fn vesicle + DNase I group;

[0079] Control group: NCM460 cells were seeded in a culture dish. When the cell density reached 60%, 200 μl PBS was added and cultured for 24 hours. Then, cell proteins were extracted according to the above steps.

[0080] Fn vesicle group: NCM460 cells were seeded in a culture dish. When the cell density reached 60%, treated Fn vesicles were added (15 μg Fn vesicles were resuspended in 200 μl PBS to obtain treated Fn vesicles) and co-cultured for 24 hours. Cell proteins were then extracted according to the above steps.

[0081] Fn vesicle + DNase I group: NCM460 cells were seeded in a culture dish. When the cell density reached 60%, 15 μg Fn vesicles treated with DNase I were added (15 μg Fn vesicles were resuspended in 200 μl PBS to obtain treated Fn vesicles, and then 50 μl of 0.01% Triton X-100 (ST1723, Biyuntian) was added to the treated Fn vesicles and incubated at room temperature for 5 minutes. Subsequently, DNase I (100 U / ml) was added and incubated at 37°C for 15 minutes to obtain 15 μg Fn vesicles treated with DNase I) and co-cultured for 24 hours, and then the cell protein was extracted according to the above-mentioned steps for extracting cell protein.

[0082] (3) Experimental results:

[0083] Figure 2 A is the Western Blot result of inflammatory factors in NCM460 cells of each group; Figure 2 B is a statistical chart of the expression of inflammatory factors in each group of NCM460 cells.

[0084] Depend on Figure 2As shown in A and B, Fn vesicles significantly increased the expression level of inflammatory factors in NCM460 cells. The expression of melanoma deficiency factor 2 (AIM2) (***p<0.001), pyrocontin D (GSDMD) (**p<0.01), pyrocontin D p30 (GSDMDp30) (***p<0.001), interleukin 18 (cleaved IL-18) (*p<0.05), caspase 1 (cleaved caspase-1) (***p<0.001), and interleukin 1β (cleaved IL-1β) (***p<0.001) in the Fn vesicle group all showed an up-regulated trend. After administration of DNase I, the expression levels of inflammatory factors AIM2 (**p<0.01), GSDMD (***p<0.001), GSDMD p30 (***p<0.001), Cleaved IL-18 (*p<0.05), Cleaved caspase-1 (***p<0.001), and Cleaved IL-1β (**p<0.01) in NCM460 cells were downregulated compared with the Fn vesicle group, indicating that the DNA of Fn vesicles is involved in the inflammatory response of intestinal epithelial cells.

[0085] VI. Construction of Animal Model

[0086] (1) Experimental animals: Six-week-old male C57BL / 6 mice were purchased from Chongqing Tengxin Biotechnology Co., Ltd.

[0087] (2) Establishment of a mouse model of ulcerative colitis combined with Fusobacterium nucleatum infection: After 7 days of adaptive feeding, 20 mice (n=20) were randomly divided into 4 groups (5 mice in each group) and given the following different treatments:

[0088] The group treated with 3% dextran sulfate sodium (DSS+Saline group): 200 μL of normal saline was given by gavage every other day for two weeks; starting from the second week, 3% dextran sulfate sodium (3% DSS) was given by drinking water for 7 days to induce ulcerative colitis;

[0089] Dextran sulfate sodium group treated with Fn vesicles (DSS+Fn vesicle group): Fn vesicles (15 μg Fn vesicles mixed with 200 μL normal saline) were given by gavage every other day for two weeks; starting from the second week, 3% dextran sulfate sodium (3% DSS) was given through drinking water for 7 days to induce ulcerative colitis;

[0090] Dextran sulfate sodium group treated with Fn vesicles and DNase I (DSS+Fn vesicles+DNase I group): DNase I-treated Fn vesicles (15 μg Fn vesicles were resuspended in 200 μl PBS to obtain treated Fn vesicles, and then 50 μl of 0.01% Triton X-100 (ST1723, Bio-Tech) was added to the treated Fn vesicles and incubated at room temperature for 5 minutes. Subsequently, DNase I (100 U / ml) was added and incubated at 37°C for 15 minutes to obtain 15 μg Fn vesicles treated with DNase I), DNase I-treated Fn vesicles were given by gavage every other day for two weeks; starting from the second week, 3% dextran sulfate sodium (3% DSS) was given through drinking water for 7 days to induce ulcerative colitis;

[0091] Dextran sulfate sodium group treated with DNase I (DSS+DNase I group): DNase I (100 U / mL, 200 μL / mouse) was administered by oral gavage every other day for two weeks; starting from the second week, 3% dextran sulfate sodium (3% DSS) was administered by drinking water for 7 days to induce ulcerative colitis;

[0092] The 3% dextran sodium sulfate in each group was freshly prepared every two days, and the actual amount of dextran sodium sulfate drunk by mice in each group was recorded every day; body weight, stool consistency and severity of blood in stool were monitored during the 3% DSS treatment, and the disease activity index (DAI) score was determined based on this; on the 8th day, the mice were killed, the colon tissue was harvested and the length was measured, and then the colon tissue was subjected to hematoxylin and eosin staining (H&E staining), immunohistochemistry staining (IHC staining), and immunofluorescence staining (IF staining).

[0093] All animal experimental procedures were approved by the Animal Ethics Committee of Chongqing Medical University (No. 2023232).

[0094] (3) Effect of DNase I on the disease activity index of mice with ulcerative colitis and Fusobacterium nucleatum infection:

[0095] Figure 3 A is the body weight of mice in each group during 3% DSS treatment, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the group treated with Fn vesicles and DNase I, and DSS+DNase I represents the group treated with DNase I; Figure 3B is the disease activity index (DAI) of each group of mice during 3% DSS treatment, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNaseI.

[0096] from Figure 3 As shown in A and B, the weight of mice in each group gradually decreased, and the disease activity index (DAI) index decreased. Compared with the DSS+Fn vesicle group, the weight loss trend of mice in the DSS+Fn vesicle+DNase I group was slower than that in the DSS+Fn vesicle group, and the disease activity index of mice in the DSS+Fn vesicle+DNase I group was lower than that in the DSS+Fn vesicle group. The above results show that the addition of DNase I can improve the weight loss and disease activity index reduction of mice with ulcerative colitis and Fusobacterium nucleatum infection.

[0097] (4) Effects of DNase I on the colon of mice with ulcerative colitis and infection with Fusobacterium nucleatum:

[0098] Figure 4 A is the colon length of mice in each group, where DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I; Figure 4 B is a comparison of the significant differences in colon length among the mice in each group, *** represents p < 0.001, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I.

[0099] from Figure 4 A shows that the colon mucosa of mice in the DSS+Saline group was smooth under naked eye observation, the colon tissue of mice in the DSS+Fn vesicle group was obviously swollen and fragile, and the swelling of the colon tissue of mice in the DSS+Fn vesicle+DNase I group was significantly reduced. Figure 4B shows that compared with the DSS+Saline group, the colon length of mice in the DSS+Fn vesicle group was significantly reduced, and compared with the DSS+Fn vesicle group, the colon length of mice in the DSS+Fn vesicle+DNase I group was significantly increased. The above results show that the addition of DNase I can improve the colon enlargement and shortening of the colon in mice with ulcerative colitis and Fusobacterium nucleatum infection.

[0100] (5) Effects of DNase I on histopathological and biochemical parameters of mice with ulcerative colitis and infection with Fusobacterium nucleatum:

[0101] Figure 5 The colon tissues of mice in each group were stained with hematoxylin and eosin (H&E staining), wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I;

[0102] Depend on Figure 5 It can be seen that compared with the DSS+Saline group, the inflammatory cell infiltration and colon structural damage shown by H&E staining of the colon tissue of mice in the DSS+Fn vesicle group were more serious, while the colon inflammatory cell infiltration and colon structural damage of mice in the DSS+Fn vesicle+DNase I group were reduced compared with the DSS+Fn vesicle group, which indicates that the addition of DNase I can alleviate the colon inflammation of mice with colitis caused by Fusobacterium nucleatum vesicles.

[0103] Figure 6 A is the immunohistochemical staining (IHC staining) of the colon tissue of each group of mice, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I; Figure 6 B is a statistical graph of the expression levels of inflammatory factors in the colon tissues of each group of mice, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I.

[0104] Depend on Figure 6A showed that compared with the DSS+Saline group, the expression levels of inflammatory factors melanoma deficiency factor 2 (AIM2) (***p<0.001), pyrocontin D (GSDMD) (***p<0.001), and interleukin 1β (IL-1β) (***p<0.001) in the colon tissue of mice in the DSS+Fn vesicle group were significantly increased, and the expression levels were statistically significant ( Figure 6 In the Fn vesicles + DNase I + DSS group, the expression levels of AIM2 (**p < 0.01), GSDMD (**p < 0.01), and IL-1β (***p < 0.001) were restored to a certain extent. The above results indicate that the addition of DNase I can improve colitis inflammation in mice with ulcerative colitis and Fusobacterium nucleatum infection.

[0105] Figure 7 A represents the expression levels of barrier factors ZO-1 and occludin in colon tissue measured by immunohistofluorescence staining (IF), wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I; Figure 7 B is a statistical graph of the expression levels of barrier factors ZO-1 and occludin in colon tissue, wherein DSS+Saline represents the group treated with 3% dextran sodium sulfate, DSS+Fn vesicles represents the dextran sodium sulfate group treated with Fn vesicles, DSS+Fn vesicles+DNase I represents the dextran sodium sulfate group treated with Fn vesicles and DNase I, and DSS+DNase I represents the dextran sodium sulfate group treated with DNase I.

[0106] Depend on Figure 7 A showed that compared with the DSS+Saline group, the expression levels of tight junction protein ZO-1 (***p<0.001) and closed protein occludin (***p<0.001) in the colon tissue of mice in the DSS+Fn vesicle group were significantly decreased, and the expression levels were statistically significant ( Figure 7 The expression levels of ZO-1 (***p<0.001) and occludin (***p<0.001) were restored to a certain extent in the DSS+Fn vesicles+DNase I group, and the results were statistically significant ( Figure 7 B). This indicates that the addition of DNase I can improve the colitis barrier damage in mice with ulcerative colitis and Fusobacterium nucleatum infection.

[0107] 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of deoxyribonuclease I in the preparation of a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection.

2. The use of deoxyribonuclease I according to claim 1 in the preparation of a drug for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The medicine for treating ulcerative colitis combined with Fusobacterium nucleatum infection is a unit preparation.

3. Use of the deoxyribonuclease I according to claim 2 in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The drug of the unit preparation has deoxyribonuclease I as an active ingredient.

4. Use of the deoxyribonuclease I according to claim 3 in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The medicine also includes auxiliary materials acceptable in pharmacy, food science or health care products science.

5. Use of the deoxyribonuclease I according to claim 1 in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The drug is in the form of an injection, an oral solution, a capsule, a tablet or a spray.

6. Use of the deoxyribonuclease I according to claim 1 in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The deoxyribonuclease I is used for preparing medicine for improving weight loss and reducing disease activity index.

7. Use of the deoxyribonuclease I according to claim 1 in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The deoxyribonuclease I is used for preparing medicine for improving colon enlargement and colon shortening.

8. Use of the deoxyribonuclease I according to claim 1 in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The deoxyribonuclease I is used to prepare a drug for reducing the expression levels of inflammatory factors AIM2, GSDMD and IL-1β and improving colitis inflammation.

9. Use of the deoxyribonuclease I according to claim 1 in the preparation of a medicament for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The deoxyribonuclease I is used to prepare a drug for increasing the expression levels of barrier factors ZO-1 and occludin and improving the barrier damage of colitis.

10. A preparation of medicine for treating ulcerative colitis combined with Fusobacterium nucleatum infection, characterized in that: The therapeutically active ingredient comprises the deoxyribonuclease I according to any one of claims 1 to 9.

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