Application of selenium-rich bifidobacterium longum fungicide in preparation of medicine for improving ulcerative colitis

Abstract

The invention discloses application of a selenium-rich bifidobacterium longum microbial inoculum in preparation of a medicine for improving ulcerative colitis, and relates to the technical field of biology. Selenium-rich bifidobacterium longum is prepared through a selenium-rich fermentation technology, inorganic nano-selenium can be converted into organic selenium, and bioavailability is improved; the strain can significantly relieve DSS-induced ulcerative colitis, reduce disease activity indexes and body weight changes, and protect intestinal barrier functions, so that ulcerative colitis is relieved, and the strain has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a selenium-enriched Bifidobacterium longum bacterial agent in preparing a medicine for improving ulcerative colitis. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease of the intestinal mucosa that includes Crohn's disease (CD) and ulcerative colitis (UC).

[0003] Commonly used IBD treatment drugs include 5-aminosalicylic acid, glucocorticoids, immunosuppressants and biological agents, but these drugs have many problems such as unsatisfactory efficacy, strong side effects and drug resistance. Therefore, despite the many treatment methods, there are still challenges in terms of efficacy and safety, and new treatment strategies need to be developed for the prevention and treatment of IBD.

[0004] Enteritis is usually associated with intestinal inflammatory response and accompanied by oxidative stress. During the pathogenesis of IBD, the content of reactive oxygen species (ROS) increases. ROS regulates cell apoptosis by oxidizing DNA, proteins, lipids and other cell structures, causing intestinal damage. Selenium is an important trace element for the human body. It plays an important role in many life processes, including anti-oxidation, enhancing immunity and scavenging free radicals. It can relieve inflammatory damage by scavenging ROS, which has potential therapeutic effects on the treatment of inflammatory bowel disease.

[0005] There is increasing evidence that intestinal flora is important for health, and flora disturbance is one of the hallmarks of IBD. Compared with healthy people, IBD patients have reduced intestinal flora diversity, reduced anti-inflammatory flora such as Firmicutes, and increased pro-inflammatory flora such as Enterobacteriaceae and Fusobacteriaceae. With the development of living standards, people prefer to alleviate IBD through non-drug means such as daily dietary supplements or functional foods. Probiotics such as Bifidobacterium and Lactobacillus are inherent intestinal bacteria that can be added to daily foods. They have good safety and are closely related to the health of the host. Among them, Bifidobacterium has many beneficial functions such as antioxidant, anti-tumor, lowering blood lipids and regulating immunity. It colonizes in the intestines shortly after the baby is born and accompanies the entire life cycle. When the healthy intestinal flora is damaged, exogenous supplementation of Bifidobacterium can quickly colonize in the intestine, which can not only enhance the intestinal barrier function, but also serve as a carrier for gene therapy or drug therapy. Through selenium-enriched culture, Bifidobacterium longum can be used to carry selenoproteins to supplement the body's selenium, effectively enhance the intestinal barrier function, alleviate inflammatory bowel disease, and reduce adverse reactions. Summary of the invention

[0006] In order to solve the problems of the prior art, the purpose of the present invention is to provide a use of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for improving ulcerative colitis.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present application provides a use of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for improving ulcerative colitis.

[0009] In a second aspect, the present application provides a probiotic preparation for improving the intestinal environment for preventing and / or treating ulcerative colitis.

[0010] In a third aspect, the present application provides a use of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and / or treating ulcerative colitis in combination with conventional immunosuppressants, small molecule drugs, traditional Chinese medicines, and biological preparations.

[0011] The first aspect of the present application provides an application of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for improving ulcerative colitis. Application of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and / or treating ulcerative colitis. Bifidobacterium longum is provided by the Industrial Microbiology Center of the China National Microbiological Culture Collection Administration, and the strain number is: ATCC 55813. The colitis is DSS-induced ulcerative colitis. The application of the selenium-enriched Bifidobacterium longum of the present invention in the prevention and / or treatment of colitis, the therapeutic effect of the selenium-enriched Bifidobacterium longum on enteritis is better than that of Bifidobacterium longum alone.

[0012] Furthermore, the selenium-enriched Bifidobacterium longum was mainly prepared from Bifidobacterium longum B. longum through a selenium-enriched fermentation process. Compared with the non-treatment group, the colon length of the selenium-enriched Bifidobacterium longum treatment group increased, the disease activity index decreased, and the stool formation was higher.

[0013] Furthermore, the content of selenium-enriched Bifidobacterium longum is not less than 1×10 8 CFU / g.

[0014] Furthermore, the fermentation medium of the selenium-enriched long bifidobacterium contains the following components: vitamin C, 4 g / L; NaCl, 2.4 g / L; Na 2 HPO 4 , 5.8g / L; KH 2 PO 4 , 0.528571g / L; glucose, 10g / L; yeast peptone, 60g / L; nano-selenium solution, 2-20mg / L.

[0015] A second aspect of the present application provides a probiotic preparation for improving the intestinal environment for preventing and / or treating ulcerative colitis, wherein the probiotic preparation comprises selenium-enriched Bifidobacterium longum.

[0016] Furthermore, the selenium-enriched Bifidobacterium longum bacterial agent includes a combination of one or more of a selenium-enriched Bifidobacterium longum fermentation culture, a fermentation supernatant, a fermentation liquid precipitate, an ultrasonic lysis supernatant, an ultrasonic lysis precipitate or a freeze-dried powder.

[0017] Furthermore, the probiotic preparation includes any one of freeze-dried powder, tablets, capsules or granules.

[0018] The third aspect of the present application provides a use of a selenium-enriched Bifidobacterium longum agent in the preparation of a drug for preventing and / or treating ulcerative colitis in combination with conventional immunosuppressants, small molecule drugs, traditional Chinese medicines, and biological preparations.

[0019] Beneficial effects: The present invention combines nano-selenium with Bifidobacterium longum and uses Bifidobacterium longum as a biological carrier for delivering selenoproteins, which has many advantages such as higher targeting, strong colonization ability, low treatment cost, convenient administration, good anti-enteritis effect, etc. The therapeutic effect of selenium-enriched Bifidobacterium longum on enteritis is better than that of Bifidobacterium longum alone.

[0020] Compared with the prior art, the present invention has the following advantages: (1) The present invention converts inorganic selenium into organic selenium through selenium-enriched fermentation, thereby improving the bioavailability and safety of selenium, making it more conducive to human absorption. Compared with other selenium sources, the conversion efficiency and delivery efficiency of selenium are improved, and the preparation method is simple and suitable for industrial production. The stable delivery of selenoproteins to the site of colon inflammation is achieved through probiotics. In addition to the probiotic function of Bifidobacterium longum, the biological activity of probiotics is also improved, so that it exhibits better effects in antibacterial, antioxidant, anti-inflammatory, and immunomodulatory aspects; its activity.

[0021] (2) The selenium-enriched Bifidobacterium longum of the present invention has the characteristic that Bifidobacterium longum can be administered orally. It has the characteristic of using Bifidobacterium longum in the preparation of drugs for preventing and / or treating ulcerative colitis in combination with conventional immunosuppressants, small molecule drugs, traditional Chinese medicines, and biological preparations. Compared with Bifidobacterium longum, the intestinal colonization ability of selenium-enriched Bifidobacterium longum remains unchanged, and it has a stronger therapeutic effect on intestinal inflammation. Selenium-enriched Bifidobacterium longum has the advantages that Bifidobacterium longum is easy to culture, low in cost, and easy to mass produce and promote application.

[0022] (3) The selenium-enriched Bifidobacterium longum of the present invention is characterized in that in a mouse acute colitis model, compared with the control strain Bifidobacterium longum, it can significantly inhibit intestinal inflammatory lesions and protect intestinal barrier function; after treatment with selenium-enriched Bifidobacterium longum, the weight of mice changes stably, and the colon length and disease activity index are close to those of healthy group mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the test examples of the present application, the drawings required for use in the test examples or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some test examples of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Description of Abbreviations

[0025] 1: healthy control group; 2: DSS non-treatment group; 3: selenium-enriched Bifidobacterium longum; 4: Bifidobacterium longum expressing endostatin; 5: Bifidobacterium longum; 6: Endostar injection.

[0026] Figure 1 The effect of the strain of the present invention on the body weight of mice with acute colitis model;

[0027] Figure 2 The effect of the strain of the present invention on the disease activity index of acute colitis model mice;

[0028] FIG3 shows the effect of the strain of the present invention on the colon length of mice with acute colitis model;

[0029] Figure 3A Representative images of the colon of mice with acute colitis model after treatment with the strain of the present invention.

[0030] Figure 3B The colon length statistics of mice with acute colitis model after treatment with the strain of the present invention.

[0031] FIG. 4 is a pathological section of the colon of mice with acute colitis model after treatment with the strain of the present invention.

[0032] Figure 4A HE staining of colon tissue of mice with acute colitis model after treatment with the strain of the present invention.

[0033] Figure 4B This is the colon tissue pathology score of acute colitis model mice after treatment with the strain of the present invention.

[0034] FIG5 shows intestinal endoscopic monitoring of acute colitis model mice during treatment with the strain of the present invention.

[0035] Figure 5A Representative images of intestinal endoscopic monitoring of acute colitis model mice during treatment with the strain of the present invention.

[0036] Figure 5B This is the scoring statistics of intestinal endoscopy monitoring of acute colitis model mice during the treatment with the strain of the present invention.

[0037] FIG6 is a graph showing the intestinal vascular permeability test of mice with acute colitis model after treatment with the strain of the present invention.

[0038] Fig. 6A Representative images of glucan permeation in colonic crypts of acute colitis model mice after treatment with the strain of the present invention.

[0039] Figure 6B The figure shows the glucan permeability statistics in the colonic crypts of mice with acute colitis model after treatment with the strain of the present invention. DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods in the following examples of the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0041] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0042] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0043] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0044] The first aspect of the embodiments of the present application provides an application of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for improving ulcerative colitis. Application of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and / or treating ulcerative colitis. Bifidobacterium longum is provided by the Industrial Microbiology Center of the China National Microbiological Culture Collection Administration, and the strain number is: ATCC55813. Colitis is DSS-induced ulcerative colitis. Application of the selenium-enriched Bifidobacterium longum of the present invention in the prevention and / or treatment of colitis, the therapeutic effect of the selenium-enriched Bifidobacterium longum on enteritis is better than that of Bifidobacterium longum alone.

[0045] In some embodiments, the selenium-enriched Bifidobacterium longum is mainly prepared by a selenium-enriched fermentation process of Bifidobacterium longum B. longum. Compared with the non-treatment group, the colon length of the selenium-enriched Bifidobacterium longum treatment group increased, the disease activity index decreased, and the stool formation was higher.

[0046] In some embodiments, the content of selenium-enriched Bifidobacterium longum is not less than 1×10 8 CFU / g.

[0047] In some embodiments, the fermentation medium of the selenium-enriched Bifidobacterium longum comprises the following components: Vitamin C, 4 g / L; NaCl, 2.4 g / L; Na 2 HPO 4 , 5.8g / L; KH 2 PO 4 , 0.528571g / L; glucose, 10g / L; yeast peptone, 60g / L; nano-selenium solution, 2-20mg / L.

[0048] A second aspect of an embodiment of the present application provides a probiotic preparation for improving the intestinal environment for preventing and / or treating ulcerative colitis, wherein the probiotic preparation includes selenium-enriched Bifidobacterium longum.

[0049] In some embodiments, the selenium-enriched Bifidobacterium longum bacterial agent includes a combination of one or more of a selenium-enriched Bifidobacterium longum fermentation culture, a fermentation supernatant, a fermentation broth precipitate, an ultrasonic lysis supernatant, an ultrasonic lysis precipitate, or a lyophilized powder.

[0050] In some embodiments, the probiotic preparation comprises any one of a lyophilized powder, a tablet, a capsule or a granule.

[0051] The third aspect of the embodiments of the present application provides a use of a selenium-enriched Bifidobacterium longum agent in the preparation of a drug for preventing and / or treating ulcerative colitis in combination with conventional immunosuppressants, small molecule drugs, traditional Chinese medicines, and biological preparations.

[0052] Example 1

[0053] The use of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for improving ulcerative colitis of the present invention is the use of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and / or treating ulcerative colitis. Bifidobacterium longum is provided by the Industrial Microbiology Center of the China Microbiological Culture Collection Administration Committee, and the strain number is: ATCC 55813. Colitis is DSS-induced ulcerative colitis. The use of the selenium-enriched Bifidobacterium longum in the prevention and / or treatment of colitis of the present invention, the therapeutic effect of the selenium-enriched Bifidobacterium longum on enteritis is better than that of Bifidobacterium longum alone.

[0054] Selenium-enriched Bifidobacterium longum is mainly prepared by a selenium-enriched fermentation process of Bifidobacterium longum. Compared with the non-treatment group, the colon length of the selenium-enriched Bifidobacterium longum treatment group increased, the disease activity index decreased, and the stool formativeness was higher.

[0055] The content of selenium-rich Bifidobacterium longum is not less than 1×10 8 CFU / g.

[0056] The fermentation medium of selenium-enriched Bifidobacterium longum contains the following components: vitamin C, 4 g / L; NaCl, 2.4 g / L; Na 2 HPO 4 , 5.8g / L; KH 2 PO 4 , 0.528571g / L; glucose, 10g / L; yeast peptone, 60g / L; nano-selenium solution, 2-20mg / L.

[0057] Preparation method of selenium-enriched Bifidobacterium longum of the present invention

[0058] (1) Prepare 3 L of Bifidobacterium longum culture medium, adjust the pH to 5.5 with NaOH, and load into a 5 L fermentation tank. The culture medium contains the following components:

[0059]

[0060] (2) Sterilize at 121°C for 20 minutes and cool to room temperature;

[0061] (3) inoculating 1 mL of thawed Bifidobacterium longum seed solution into 200 mL of culture medium and performing activation culture at 40°C for 24 hours;

[0062] (4) The activated Bifidobacterium longum seed liquid was transferred to 1 L of culture medium at an inoculum volume of 6%, and the nano-selenium solution filtered through a 0.22 μm filter membrane was added to a final concentration of 2-20 mg / L (the preparation method refers to the patents applied for: a method for preparing a nano-selenium solution, application number: 2024110944130; a selenium-enriched Bifidobacterium longum agent and its preparation method, application number: 2024110944126).

[0063] (5) The fermentation was continued for 9 hours, during which nitrogen was passed to remove air, and 30% glucose was added at a constant rate of 50 mL / h to obtain a selenium-enriched Bifidobacterium longum inoculum.

[0064] Example 2

[0065] The invention discloses a probiotic preparation for improving intestinal environment and for preventing and / or treating ulcerative colitis. The probiotic preparation comprises selenium-enriched Bifidobacterium longum.

[0066] The selenium-enriched Bifidobacterium longum bacterial agent comprises a combination of one or more of a selenium-enriched Bifidobacterium longum fermentation culture, a fermentation supernatant, a fermentation liquid precipitate, an ultrasonic lysis supernatant, an ultrasonic lysis precipitate or a freeze-dried powder.

[0067] Example 3

[0068] The difference between Example 3 and Example 2 is that the selenium-enriched Bifidobacterium longum bacterial agent is a fermented culture of selenium-enriched Bifidobacterium longum and the probiotic preparation is a freeze-dried powder.

[0069] Example 4

[0070] The difference between Example 4 and Example 2 is that the selenium-enriched Bifidobacterium longum bacterial agent is a combination of four of fermentation supernatant, fermentation broth precipitate, ultrasonic lysis supernatant, ultrasonic lysis precipitate or freeze-dried powder. The probiotic preparation is a tablet.

[0071] Example 5

[0072] The difference between Example 5 and Example 2 is that the selenium-enriched Bifidobacterium longum bacterial agent is a fermentation broth precipitate and the probiotic preparation is a capsule.

[0073] Example 6

[0074] The difference between Example 6 and Example 2 is that the selenium-enriched Bifidobacterium longum bacterial agent is ultrasonic lysis precipitation and the probiotic preparation is granules.

[0075] Example 7

[0076] The invention discloses an application of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and / or treating ulcerative colitis in combination with conventional immunosuppressants, small molecule drugs, traditional Chinese medicines and biological preparations.

[0077] Test Example 1

[0078] The detection methods involved in the following embodiments are as follows:

[0079] Disease Activity Index (DAI) detection method:

[0080] The disease activity index is scored by three aspects: weight loss, stool shape and blood in the stool (the specific scoring criteria are shown in Table 1). During the mouse modeling and treatment period, the mouse weight, stool shape and blood in the stool were observed and recorded every day, and scored according to Table 1, DAI = (weight loss score + stool shape score + blood in the stool degree score) / 3. Weight loss is divided into 5 levels: 0%, 1-5%, 6-10%, 11-15%, >15%; stool shape is divided into 5 levels: normal, soft, loose, unshaped, and loose stools; blood in the stool is divided into 4 levels: normal, a small amount of blood, a large amount of blood, and blood in the stool visible to the naked eye. The disease activity index scoring criteria are shown in Table 1:

[0081] Table 1

[0082] Fraction Weight loss Stool shape Blood in stool 0 0％ normal normal 1 1-5％ Softer 2 6-10％ Loose Small amount of blood 3 11-15％ No shape Large amounts of blood 4 ＞15％ Loose stools Naked bloody stool

[0083] The tissue injury pathological scoring criteria are as follows:

[0084] Tissue injury pathology scoring was performed on the HE-stained sections of the colon tissue of each group of mice based on three aspects: severity of inflammation, depth of lesions, and infiltration of inflammatory cells (the specific scoring criteria are shown in Table 2). The scoring was performed according to Table 2, and the tissue injury score = inflammation score + lesion depth score + inflammatory cell infiltration score. The severity of inflammation is divided into 4 levels: none, mild, moderate, and severe; the depth of lesions is divided into 4 levels: none, mucosal layer, submucosal layer, and transintestinal wall injury; the infiltration of inflammatory cells is divided into 4 levels: no or a small amount of inflammatory cells in the mucosal lamina propria, more inflammatory cells in the mucosal lamina propria, inflammatory cells spread to the submucosal layer, and inflammatory cells infiltrated throughout the entire layer. The tissue injury pathology scores are shown in Table 2:

[0085] Table 2

[0086]

[0087]

[0088] The UCEIS scoring criteria are as follows:

[0089] The endoscopic severity index is scored by three aspects: vascular texture, bleeding, erosion and ulcer (the specific scoring criteria are shown in Table 3). During the treatment of mice, intestinal lesions were monitored by endoscopy and scored according to Table 3. The endoscopic severity index score = vascular texture score + bleeding score + erosion and ulcer score. Vascular texture is divided into 3 levels: normal, patchy disappearance, and complete disappearance; bleeding is divided into 4 levels: none, mucosal bleeding, mild bleeding in the intestinal cavity, and severe bleeding in the intestinal cavity; erosion and ulcer conditions are divided into 4 levels: none, erosion, superficial ulcer, and deep ulcer. The endoscopic severity index score is shown in Table 3:

[0090] Table 3

[0091] Fraction Blood vessel texture Bleeding Erosions and ulcers 0 normal none none 1 Disappearance of plaque Mucosal bleeding erosion 2 disappear completely Mild bleeding in the intestinal lumen Superficial ulcer 3 Severe intestinal bleeding Deep ulcer

[0092] Test Example 2

[0093] Effect of selenium-enriched Bifidobacterium longum on the symptoms of DSS-induced acute colitis in mice

[0094] The selenium-enriched Bifidobacterium longum in the present invention is obtained by fermentation of Bifidobacterium longum B. longum and its derivatives or genetically modified strains, and its intestinal colonization ability remains unchanged. The following only takes Bifidobacterium longum as an example.

[0095] Experimental grouping: 32 SPF-grade 6-8-week-old (19±1g) female C57BL / 6 mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. They were kept in an environment with an ambient temperature of 25±2℃ and a humidity of 50±5%, with 12h of light and dark alternation every day, free access to sterilized water, and adaptive feeding for 7 days. They were randomly divided into 4 groups, with 8 mice in each group; the 4 groups were 1: healthy control group; 2: DSS non-treatment group; 3: selenium-enriched Bifidobacterium longum; 4: Bifidobacterium longum expressing endostatin (positive control group, a strain that has been shown to have significant enteritis treatment effects in previous studies in this laboratory, expressing recombinant human endostatin protein through plasmid pBV222 plasmid; Frontiers in Microbiology, 2022, 13: 927277.);

[0096] 3% DSS solution: 9 g of dextran sulfate sodium (DSS) was dissolved in 300 mL of sterile water to prepare a DSS solution with a concentration of 3% (w / v).

[0097] Treatment regimen: During the modeling period, the healthy control group (group 1) freely drank sterilized water, and the non-treatment group and treatment group (groups 2-4) freely drank 3% DSS solution; the healthy control group and non-treatment group (groups 1 and 2) were gavaged with 200 μL of sterile PBS buffer every day, and the selenium-enriched Bifidobacterium longum and endostatin-expressing Bifidobacterium longum treatment groups (groups 3 and 4) were gavaged with 200 μL of selenium-enriched Bifidobacterium longum or endostatin-expressing Bifidobacterium longum bacterial solution every day (selenium-enriched Bifidobacterium longum or endostatin-expressing Bifidobacterium longum was resuspended in sterile PBS buffer, 1.5×10 10 CFU / kg); the mice in each group were weighed daily, and the disease activity index (DAI, Table 1) of each group of mice was observed and recorded. After the experiment, the mice were anesthetized and killed, and the entire colon (from the end of the cecum to the anus) was taken and placed in pre-cooled PBS on ice. The colon length was then measured and representative pictures were taken.

[0098] During the modeling period, the weight of mice in the healthy control group was stable and did not decrease significantly. The weight of mice in the DSS non-treatment group began to drop rapidly on the 4th day, and by the end of the experiment, the average weight loss was 25.5%. In contrast, the weight fluctuation of mice in the selenium-enriched longum Bifidobacterium treatment group was reduced, with an average weight loss of 17.7%. The weight of mice in the longum Bifidobacterium treatment group expressing endostatin was relatively stable, and the average weight loss was only 4.2% at the end of the experiment, which was significantly better than the DSS group ( Figure 1 ).

[0099] During the modeling period, along with weight loss, the mice in the DSS non-treatment group showed symptoms such as reduced stool formation and blood in the stool after the 4th day, proving that DSS induced ulcerative colitis. At the end of treatment, the average DAI of the mice in the DSS non-treatment group increased to 3.4. The enteritis in the group treated with selenium-enriched Bifidobacterium longum was alleviated, with high stool formation and reduced blood in the stool. The average DAI of the mice was 2.8. The stool formation and blood in the stool of the mice in the group treated with Bifidobacterium longum expressing endostatin were not serious, and enteritis progressed slowly. At the end of treatment, the average DAI was only 1.5, showing a good enteritis inhibition effect ( Figure 2 ).

[0100] At the end of the experiment, the colon lengths of mice in each group were counted. The average colon length of mice in the healthy control group was 7.3 cm, and the feces in the intestine were highly formed. The colons of mice in the DSS non-treatment group were significantly shortened, with an average length of 5.5 cm, which was 24.1% shorter than that of normal mice. The intestines were pink, with no formed feces, and the degree of ulcerative enteritis was severe. Treatment with selenium-enriched Bifidobacterium longum alleviated the atrophy of the colon length, with an average length of 5.7 cm. Among them, Bifidobacterium longum expressing endostatin had the best treatment effect, with a colon length of 6.7 cm, close to that of healthy mice, and no obvious inflammatory areas in the intestines, and the feces were highly formed ( Figure 3A , 3B).

[0101] The above results show that selenium-enriched Bifidobacterium longum can alleviate the symptoms of inflammatory bowel disease in mice. The selenium-enriched Bifidobacterium longum of the present invention can further enhance the efficacy of inhibiting enteritis through subsequent genetic engineering modification.

[0102] Test Example 3

[0103] Selenium-enriched Bifidobacterium longum improves pathological damage in mice with ulcerative colitis

[0104] After the mice were anesthetized and killed, the whole colon (from the end of the cecum to the anus) was dissected out, the length was measured and representative pictures were taken. 0.5-1cm long colon tissue was cut at 1cm from the end of the cecum, the fecal residue inside the colon was washed clean with PBS buffer, and the outside of the colon was trimmed flat with a surgical blade. The trimmed colon tissue was soaked in 4% paraformaldehyde solution for more than 24 hours and then soaked in ethanol for dehydration: 70% ethanol soaked overnight; 80% ethanol soaked for 1h; 95% ethanol soaked for 30min, replaced with 95% ethanol, and soaked again for 30min; anhydrous ethanol soaked for 15min, replaced with anhydrous ethanol, and soaked again for 15min; xylene soaked for 15min, replaced with xylene, and soaked again for 15min; 65℃ soaked in paraffin for 1h. The tissue soaked in paraffin was embedded in a paraffin embedding machine. First, add the melted paraffin into the embedding frame. Before the paraffin solidifies, fix the colon cross section upward in the embedding frame and place it on a -20℃ freezer to cool. After the paraffin block cools and solidifies, remove the wax block, trim it, and store it in a 4℃ refrigerator.

[0105] Paraffin sectioning: Fix the trimmed and cooled paraffin block on the paraffin slicer, install the blade, and adjust the blade and the wax block so that the two are in contact. Adjust the thickness of the cut wax to 15-20μm, quickly rotate the slicer wheel, and trim the surface of the tissue. Adjust the slice thickness to 3-5μm and cut out a complete wax slice. Float the slice in cold water, and transfer it to 42℃ hot water after unfolding, facing up. After the slice is fully unfolded, pick it up and attach it to the adhesive slide, and let it stand overnight at room temperature to dry.

[0106] HE staining: Place the tissue slide in a 60℃ constant temperature oven for 2-3 hours before dewaxing. Take out the slide, soak in xylene for 15 minutes, replace xylene, and soak again for 15 minutes; soak in anhydrous ethanol for 5 minutes; soak in 95% ethanol for 3 minutes; soak in 80% ethanol for 1 minute; soak in pure water for 3 minutes, replace pure water, and soak again for 2 minutes; fully immerse the tissue area on the slide in hematoxylin staining solution for 5-10 minutes, and rinse the excess stain with pure water. Immerse in anhydrous ethanol containing 1% hydrochloric acid for 3 seconds to lighten the color of hematoxylin. Immerse in 1% NH 3 H 2 O solution turns blue for 2-3 minutes to make the hematoxylin color darker. After rinsing with pure water, observe under a microscope and adjust according to the color depth of hematoxylin. If the color is too dark, soak it again in hydrochloric acid ethanol. If the color is too light, re-stain it with hematoxylin. When the hematoxylin color is appropriate, soak it in eosin staining solution for 10 seconds and rinse the excess stain with pure water. Soak in 95% ethanol for 2 minutes; soak in anhydrous ethanol for 2 minutes; soak in xylene for 5 minutes, replace xylene, and soak again for 5 minutes; use neutral resin to seal the slide and store at room temperature.

[0107] The prepared colon tissue HE-stained sections were scanned using a Leica THUNDER microscopy system, and the tissue damage was pathologically scored according to the degree of inflammation, lesion depth, and inflammatory cell infiltration in the tissue sections (Table 2).

[0108] Representative images of HE staining of colon sections showed that the colon structure of mice in the healthy group was clearly layered, the glands were closely arranged and abundant, the mucosal epithelial tissue was intact, the cell morphology was normal, there was no obvious inflammatory cell infiltration, and no obvious abnormalities were observed; in contrast, the DSS-untreated group showed extensive ulcerative lesions, fibrous tissue hyperplasia, mucosal epithelial damage, edema in the submucosal layer and muscular layer, a large number of inflammatory cell infiltrations, disappearance of glandular structure, and infiltration of inflammatory exudates in the intestinal cavity; after treatment with selenium-enriched long bifidobacterium, inflammatory lesions were alleviated, inflammatory cell infiltration was reduced, glandular structure was normal, and there was a small amount of floc in the intestinal cavity. No obvious ulcers were observed in the colon sections of the group treated with long bifidobacterium expressing endostatin, the cell morphology was intact, there was no obvious inflammatory cell infiltration, and the tissue was similar to that of healthy mice ( Figure 4A ).

[0109] The histopathological score was evaluated according to the criteria in Table 2. The results showed that the intestinal damage in the DSS group was severe, with a tissue damage score of 8.3 points. The score of the selenium-enriched longum Bifidobacterium treatment group was significantly reduced to 6.3 points. The longum Bifidobacterium expressing endostatin had the best treatment effect, with a score of only 3.3 points, which was close to the healthy control group ( Figure 4B ).

[0110] The above results indicate that selenium-enriched Bifidobacterium longum treatment can reduce intestinal tissue pathological damage caused by ulcerative colitis in mice and enhance the inflammatory resistance of intestinal tissue.

[0111] Test Example 4

[0112] Alleviating effect of selenium-enriched Bifidobacterium longum on colonic inflammation in mice with ulcerative colitis

[0113] Experimental groups: 30 SPF-grade 6-8-week-old (19±1g) female C57BL / 6 mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. They were kept in an environment with an ambient temperature of 25±2℃ and a humidity of 50±5%, with 12h of light and dark alternation every day, free access to sterilized water, and adaptive feeding for 7 days. They were randomly divided into 6 groups, 5 mice in each group; the 6 groups were 1: healthy control group; 2: DSS non-treatment group; 3: selenium-enriched Bifidobacterium longum; 4: Bifidobacterium longum expressing endostatin; 5: Bifidobacterium longum; 6: Endostatin injection (recombinant human endostatin).

[0114] During the modeling period, the healthy control group (group 1) freely drank sterilized water, and the non-treatment group and treatment group (groups 2-6) freely drank 3% DSS solution; the healthy control group and non-treatment group (groups 1 and 2) were gavaged with 200 μL of sterile PBS buffer every day. The probiotic treatment groups (groups 3, 4 and 5) were gavaged with 200 μL of probiotic solution (selenium-enriched Bifidobacterium longum, Bifidobacterium longum expressing endostatin or Bifidobacterium longum resuspended in sterile PBS buffer, 1.5×10 10 CFU / kg). The Endo treatment group was intraperitoneally injected with 100 μL of recombinant human endostatin injection (3 mg / kg, Endo, Innovent Biologics). The mice were weighed daily, and the feces formation and blood in the stool were observed. After drinking DSS solution for six consecutive days, the colon inflammation and ulceration of the mice were observed by animal electronic endoscopy. The endoscopic enteritis severity index was scored based on the vascular texture, bleeding, erosion and ulceration.

[0115] After anesthesia, the mouse is fixed to the laboratory table with medical tape for colonoscopy. After confirming that all parts of the endoscope are connected, gently apply medical silicone oil to the observation window and lighting window at the end of the endoscope head. Turn on the power of the endoscope image processor, adjust the image white balance and brightness to achieve a suitable image effect for observation. According to the intestinal contents, air and water are supplied, and the angle handle is operated to adjust the bend angle. The head of the animal endoscope is slowly inserted 1-2cm into the colon through the anus while observing. Clean the mucus on the mirror surface by supplying water, air and suction to ensure a clear picture. After the test is completed, use 2% glutaraldehyde disinfectant to clean and disinfect the endoscope.

[0116] According to the endoscopic monitoring images, the inner wall of the colon of healthy mice was smooth, the blood vessels were clear and complete, and there were no symptoms such as ulcers and inflammatory bleeding. In contrast, the DSS non-treatment group had severe intestinal inflammatory damage, extensive deep ulcers on the inner wall of the intestine, hyperplasia and bulging of the mucosal epithelium, compression of the intestine, disappearance of vascular texture, bleeding in the intestinal cavity accompanied by a large amount of inflammatory exudate. The ulcers in the Bifidobacterium longum (5 groups) treatment group were significantly relieved, and there was no intestinal bleeding. The treatment effect of selenium-enriched Bifidobacterium longum (3 groups) was increased, a small amount of vascular texture was visible, and the intestinal exudate was significantly reduced, and there was no bleeding, indicating that ulcerative enteritis was relieved. The treatment groups of Bifidobacterium longum (4 groups) and Endo (6 groups) expressing endostatin had the best treatment effect. The inner wall of the colon was smooth, obvious vascular texture was visible, and the number of vascular veins was less than that of the healthy control group. There was no obvious bleeding and ulcers on the inner wall of the intestine ( Figure 5A ).

[0117] The endoscopic severity index UCEIS (ulcerative colitis endoscopic severity index scoring system) score was evaluated according to the criteria in Table 3. The results showed that the colon damage in the non-DSS treatment group was severe, with an endoscopic score of 13. Bifidobacterium longum treatment can relieve intestinal inflammation, reducing the endoscopic score to 8.4. Through targeted delivery of selenoproteins, the selenium-enriched Bifidobacterium longum treatment group effectively reduced the degree of intestinal ulcers, with an endoscopic score of 7.2, achieving better therapeutic effects. The endoscopic score of intraperitoneal injection of recombinant human endostatin was 4.2, proving that endostatin has a good therapeutic effect on DSS-induced ulcerative colitis. The Bifidobacterium longum expressing endostatin had the best therapeutic effect, with an endoscopic score of 3.6, and the intestinal state was greatly improved. ( Figure 5B ).

[0118] The above results show that probiotic treatment can reduce intestinal ulcerative lesions caused by ulcerative colitis in mice. By delivering selenoproteins through selenium-enriched fermentation, selenium-enriched Bifidobacterium longum can achieve a better therapeutic effect than Bifidobacterium longum. Bifidobacterium longum expressing endostatin has the best therapeutic effect, indicating that endostatin can protect the intestinal barrier and reduce mucosal damage in an inflammatory environment.

[0119] Test Example 5

[0120] Protective effect of selenium-enriched Bifidobacterium longum on colonic vascular permeability in mice with ulcerative colitis

[0121] The specific grouping and feeding and administration methods are the same as the grouping and modeling steps in Example 3.

[0122] Sample processing: Before the end of the experiment, 70kDa FITC-dextran (10mg / mL) was injected through the tail vein, 100μL per mouse, and the mice were anesthetized and killed 30min later. The whole colon (from the end of the cecum to the anus) was dissected out, and 1-2cm of colon tissue was cut 1cm from the end of the cecum, and the edges of about 2mm at both ends were cut off. The intestine was cut longitudinally, with the inside facing up, placed on a slide, and sealed with anti-fluorescence quenching sealing agent.

[0123] The Leica THUNDER microscope was used to observe the FITC fluorescence signal in the colon samples. The results showed that the blood vessels of healthy mice were intact, with clear veins and no fluorescence signals in the intestinal crypts. However, the blood vessels of mice in the DSS non-treatment group had poor continuity, and the surrounding FITC-dextran diffused and accumulated in the intestinal crypts, indicating that intestinal inflammation caused increased vascular permeability and impaired intestinal barrier function, leading to the exudation of inflammatory substances and the infiltration of inflammatory cells, further exacerbating inflammatory damage ( Fig. 6A ), the vascular permeability was calculated by the ratio of FITC signal in the crypt to the total signal. The vascular permeability of mice in the DSS non-treatment group was 23.6%, which was significantly higher than that of healthy mice ( Figure 6B). Bifidobacterium longum treatment can alleviate the symptoms of DSS-induced ulcerative colitis, so the intestinal barrier function is restored and vascular permeability is reduced to 15.7%. The delivery of selenoproteins through selenium-enriched fermentation can reduce oxidative damage at the inflammatory site and reduce vascular permeability to 10.7%, which is close to the endostatin treatment group. Endostatin treatment can inhibit the formation of intestinal angiogenesis under inflammatory conditions, thereby reducing the infiltration of inflammatory cells and the exudation of inflammatory substances, and enhancing the barrier function of the intestine. Therefore, the intestinal vascular permeability of mice in the Bifidobacterium longum and Endo treatment groups expressing endostatin was low, and the mucosal and glandular structures were intact and tightly arranged ( Figure 4A ), the vascular veins were clearly visible, the barrier function was intact, and the vascular permeability was only 7.6% and 7.5%, which was close to the healthy control group (4.0%).

[0124] The above results indicate that selenium-enriched Bifidobacterium longum can improve the permeability of intestinal vascular in mice with ulcerative colitis, achieving an effect close to that of endostatin, and enhance the intestinal barrier function.

[0125] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above test examples, and the above test examples and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. Use of a selenium-enriched Bifidobacterium longum bacterial agent in the preparation of a drug for improving ulcerative colitis.

2. The use according to claim 1, characterized in that: The selenium-enriched Bifidobacterium longum bacterial agent is prepared from Bifidobacterium longum B. longum through a selenium-enriched fermentation process.

3. The use according to claim 1, characterized in that: The content of selenium-rich Bifidobacterium longum is not less than 1×10 8 CFU / g.

4. The use according to claim 3, characterized in that: The fermentation medium of the selenium-enriched long bifidobacterium comprises the following components: vitamin C, 4 g / L; NaCl, 2.4 g / L; Na2HPO4, 5.8 g / L; KH2PO4, 0.528571 g / L; glucose, 10 g / L; yeast peptone, 60 g / L; and nano-selenium solution, 2-20 mg / L.

5. A probiotic preparation for improving the intestinal environment for preventing and / or treating ulcerative colitis, characterized in that: The probiotic preparation comprises selenium-enriched Bifidobacterium longum.

6. The probiotic preparation according to claim 1, characterized in that: The selenium-enriched Bifidobacterium longum bacterial agent comprises a combination of one or more of selenium-enriched Bifidobacterium longum fermentation culture, fermentation supernatant, fermentation liquid precipitate, ultrasonic lysis supernatant, ultrasonic lysis precipitate or freeze-dried powder.

7. The probiotic preparation according to claim 5, characterized in that: The probiotic preparation includes any one of freeze-dried powder, tablets, capsules or granules.

8. Use of the selenium-enriched Bifidobacterium longum agent according to claim 1 in the preparation of a drug for preventing and / or treating ulcerative colitis in combination with conventional immunosuppressants, small molecule drugs, traditional Chinese medicines, and biological agents.

Images