Application of probiotics Blauria wexlerae in improvement or treatment of children inflammatory bowel disease

Abstract

The invention discloses an application of probiotics Blauria winteriae in the preparation of a medicine or food for improving or treating the inflammatory bowel disease of children, and relates to the field of medicines or foods for improving or treating the inflammatory bowel disease of children. According to the invention, the probiotic Blauria wexlerae is used as an effective component to be prepared into medically and food acceptable dosage forms, so that the children inflammatory bowel disease can be obviously improved or treated, and the application value of the probiotic is improved.

Description

Technical Field

[0001] The present invention relates to the field of probiotic drugs, in particular to the technical field of probiotics for treating inflammatory bowel disease in children, specifically to the use of probiotics Blautia wexlerae in improving or treating inflammatory bowel disease in children. Background Art

[0002] Inflammatory bowel disease (IBD) refers to a group of nonspecific chronic gastrointestinal inflammatory diseases of unknown cause, mainly including Crohn's disease (CD) and ulcerative colitis (UC). Childhood CD can occur in any part of the gastrointestinal tract, and usually develops between the ages of 9 and 17, with symptoms of abdominal pain, diarrhea, weight loss, and may be accompanied by growth retardation, abdominal masses, intestinal fistulas, perianal lesions, fever, anemia and other systemic manifestations. Childhood UC is a continuous chronic inflammation of the colonic mucosa, often starting from the rectum and gradually involving the entire colon. It is more common in children over 6 years old, and is often manifested as persistent bloody stools with diarrhea, which may be accompanied by malnutrition, joints, skin, eyes, mouth and liver and gallbladder and other extraintestinal manifestations. IBD is a protracted disease with a long course, which causes physical and mental burdens to children. The treatment drugs are expensive, and the medical and economic burdens on society and families are heavy. The incidence of IBD is about 30-130 / 100,000 people in North America, Oceania and most European countries. In the past decade, the incidence and prevalence of IBD in emerging industrialized countries in Africa, Asia, and South America have gradually increased. The incidence of IBD in children in Shanghai has increased from 0.05 / 100,000 in 2001 to 0.61 / 100,000 in 2010, an increase of 1,220%. Compared with adults, the most notable feature of IBD in children is slow growth and development and significantly fewer treatment options than adults.

[0003] At present, the main treatment options for inflammatory bowel disease in children include nutritional therapy, drug therapy and surgical treatment. Nutritional therapy includes enteral nutrition, parenteral nutrition, and vitamin and trace element supplementation; drug therapy mainly includes aminosalicylic acid preparations, glucocorticoids, immunosuppressants and biological agents; surgical intervention is required when children have surgical indications such as intestinal obstruction and intestinal perforation. However, the existing treatment options have different effects on individuals. Some children eventually develop severe IBD due to ineffective drug treatment, and even require surgical intervention, which seriously affects the quality of life of children. Therefore, new treatment options are urgently needed to improve the prognosis of children with IBD. At present, the causal relationship between intestinal flora dysbiosis and IBD has not been fully confirmed, but many studies have found that at least some changes in the flora are the cause of IBD or play a protective role in IBD.

[0004] Our previous clinical studies have found that the content of Blautia wexlerae in the feces of healthy children is significantly higher than that of children with IBD. Studies have shown that Blautia wexlerae isolated from the human intestine has been shown to have the potential to alleviate obesity and type 2 diabetes, and is expected to become the next generation of star probiotics. Unlike other next-generation probiotic candidates, such as Oscillospira (which cannot be purified, which greatly limits the research and further development of this microorganism), Blautia wexlerae can be purified, allowing it to quickly carry out preclinical and clinical studies. Blautia wexlerae can be used as a functional food or special medical food for metabolic diseases. Therefore, we infer that Blautia wexlerae may play an important role in protecting the occurrence and development of IBD. Animal experiments were conducted using Blautia wexlerae. The experimental results showed that the bacteria can significantly improve dextran sulfate sodium (DSS)-induced colitis in 3-week-old mice in both active and inactivated states. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is the use of a probiotic Blautiawexlerae in the preparation of a medicine or food for improving or treating inflammatory bowel disease in children. The probiotic Blautiawexlerae is used as an active ingredient to prepare a dosage form acceptable in medicine and food, which can significantly improve or treat inflammatory bowel disease in children and improve the application value of the probiotic.

[0006] To achieve the above object, the present invention provides a use of a probiotic Blautia wexlerae in the preparation of a medicine or food for improving or treating inflammatory bowel disease in children.

[0007] The probiotics are Blautia wexlerae, with strain number: DSM19850.

[0008] Preferably, the medicine or food uses the probiotic Blautia wexlerae as an active ingredient and is prepared into a dosage form acceptable in medicine or food.

[0009] Preferably, the dosage form includes powder, tablet, granule, capsule or drink.

[0010] Preferably, the probiotic Blautia wexlerae is an inflammatory factor inhibitor.

[0011] Preferably, the probiotic Blautia wexlerae is in an active state or inactivated by heat treatment.

[0012] Another aspect of the present invention provides a drug for improving or treating inflammatory bowel disease in children, the drug comprising an effective amount of probiotics Blautia wexlerae and medically acceptable excipients.

[0013] Preferably, the effective dose of the probiotic Blautia wexlerae is 1*10^9 CFU / day.

[0014] The present invention also provides a food for improving or treating inflammatory bowel disease in children, wherein the food comprises an effective amount of probiotics Blautia wexlerae and auxiliary materials acceptable to food.

[0015] Preferably, the effective amount of the probiotic Blautia wexlerae is 1*10^9 CFU / day.

[0016] Beneficial technical effects of the present invention:

[0017] 1. The present invention provides a use of probiotics Blautia wexlerae in the preparation of medicines or foods for improving or treating inflammatory bowel disease in children. Probiotics Blautia wexlerae are used as active ingredients to prepare a dosage form acceptable in medicine and food, which can significantly improve weight loss, immune factor imbalance, increased oxygen free radicals and intestinal flora disorders caused by inflammatory bowel disease in children.

[0018] 2. The present invention uses induction of colitis in mice to intervene in the administration of drugs or food. Compared with the model group, the weight loss was significantly improved; the disease activity index of mice was reduced; the level of serum inflammatory factors was reduced; the proportion of beneficial bacteria in the intestines of mice was increased, and the proportion of harmful bacteria was reduced. It is shown that the drugs or foods prepared by the probiotics Blautia wexlerae provided by the present invention can improve inflammatory bowel disease in children caused by various factors.

[0019] 3. The Blautia wexlerae of the present invention has obvious effect in improving colon inflammation in colitis mice, has low cost, and has good clinical application prospects; the Blautia wexlerae of the present invention can be used as an inflammatory factor inhibitor for basic research on IBD; the Blautia wexlerae of the present invention can obviously improve colon inflammation in mice both in an active state and after inactivation by heat treatment; the animal model of the present invention is established in young mice, and the mice have not reached adult age after the modeling is completed, which is more in line with the clinical onset age of IBD in children.

[0020] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is that Blautia wexlerae (denoted as B. wexlerae in the attached figure) after oral administration improves the weight gain and intestinal inflammation of mice with DSS-induced colitis;

[0022] Among them: (A) Line graph of weight changes of mice in the Control group (n=10), DSS group (n=10), DSS+Blautia wexlerae group (n=10), DSS+inactivated Blautia wexlerae group (n=8) and Blautia wexlerae group (n=5); (B) Final weight changes of mice in the five groups; (C) Comparison of disease activity scores of mice in the five groups; (D) Comparison of the ratio of spleen weight to body weight of mice in the five groups; (E) Comparison of colon length of mice in the five groups; (F) Comparison of the ratio of colon weight to colon length of mice in the five groups. (*P<0.05, ***P<0.001, ****P<0.0001);

[0023] Figure 2 Blautia wexlerae improved colon inflammation in mice with DSS-induced colitis after oral administration;

[0024] Among them: (A) The gross appearance of the colon of mice in the Control group, DSS group, DSS+Blautia wexlerae group, DSS+inactivatedBlautia wexlerae group, and Blautia wexlerae group; (B) H&E staining of the colon tissue of mice in the Control group, DSS group, DSS+Blautia wexlerae group, and DSS+inactivated Blautia wexlerae group, as well as the pathological scores of the colon tissue of mice in the four groups. (****P<0.0001);

[0025] Figure 3 It is that Blautia wexlerae after oral gavage improves the serum inflammatory level of mice with DSS-induced colitis;

[0026] Among them: (A) Comparison of fecal calprotectin levels in mice in the Control group, DSS group and DSS+Blautia wexlerae group; (B) Comparison of CRP levels in the three groups of mice; (C) Comparison of serum IL-1β levels in the three groups of mice; (D) Comparison of serum IL-2 levels in the three groups of mice; (E) Comparison of serum IL-6 levels in the three groups of mice; (F) Comparison of serum IL-9 levels in the three groups of mice; (G) Comparison of serum IL-13 levels in the three groups of mice; (H) Comparison of serum IL-17A levels in the three groups of mice; (I) Comparison of serum IL-18 levels in the three groups of mice; (J) Comparison of serum IL-22 levels in the three groups of mice; (K) Comparison of serum IL-23 levels in the three groups of mice; (L) Comparison of serum TNF-alpha levels in the three groups of mice. Control group n=6-10, DSS group n=3-10, DSS+Blautia wexlerae group n=3-5. (*P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0027] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0028] In the drawings, components with the same structure are indicated by the same numerical reference numerals, and components with similar structures or functions are indicated by similar numerical reference numerals. The size and thickness of each component shown in the drawings are arbitrarily shown, and the present invention does not limit the size and thickness of each component. In order to make the illustration clearer, the thickness of the components is appropriately exaggerated in some places in the drawings.

[0029] The embodiments of the present invention are described in detail below. It should be noted that the embodiments are descriptive rather than restrictive and cannot be used to limit the protection scope of the present invention.

[0030] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0031] The present invention provides an application of probiotics Blautia wexlerae in the preparation of medicines or foods for improving or treating inflammatory bowel disease in children. Using probiotics Blautia wexlerae as an effective ingredient to prepare a dosage form acceptable in medicine and food can significantly improve weight loss, immune factor imbalance, increased oxygen free radicals and intestinal flora disorder caused by inflammatory bowel disease in children.

[0032] In order to achieve this object, the technical solution of the present invention is as follows:

[0033] The present invention provides a probiotic for improving IBD in children, wherein the probiotic is Blautia wexlerae, with a strain number of DSM19850. In the present invention, it was found that Blautia wexlerae can improve colon inflammation in DSS-induced colitis mice and reduce the expression level of inflammatory factors. IBD mouse modeling was performed by feeding DSS drinking water to 3-week-old wild-type (background strain: C57BL / 6J) male mice that had just been weaned, and by gavage of Blautia wexlerae to DSS mice, the changes in mouse weight, diarrhea, and bloody stools were observed, and the changes in mouse colon inflammation, colon pathological changes, and serum inflammatory factor levels were detected to evaluate the improvement effect of Blautia wexlerae on DSS mice.

[0034] In the present invention, a Control group, a DSS group, a DSS+Blautia wexlerae gavage group (DSS+Blautia wexlerae group), a DSS+inactivated Blautia wexlerae gavage group (DSS+inactivated Blautia wexlerae group), and a Blautia wexlerae gavage group (Blautia wexlerae group) were set.

[0035] In the present invention, the control group was given normal drinking water and normal diet for 9 consecutive days.

[0036] In the present invention, the DSS group was given 3.0% DSS in drinking water and a normal diet for 9 consecutive days.

[0037] In the present invention, the Blautia wexlerae gavage method is to gavage Blautia wexlerae starting from the first day of the experiment and continuing for 9 days.

[0038] In the present invention, the method of administering bacteria is to infuse Blautia wexlerae into the stomach of mice via a gavage needle.

[0039] In the present invention, the intragastric dose of Blautia wexlerae is 1*10^9 CFU per mouse each time, dissolved in PBS, with a total volume of 200ul.

[0040] In the present invention, the heat-treated inactivated Blautia wexlerae is Blautia wexlerae inactivated at a high temperature of 121° C. for 15 minutes.

[0041] In the present invention, the frequency of intragastric administration of Blautia wexlerae is once a day, starting from the first day of the experiment, and the administration is continued for 9 days.

[0042] In the present invention, the breeding environment is SPF grade, with constant temperature (22-26° C.), constant humidity (45-55%), and a 12-hour day and night cycle.

[0043] In the method of the present invention, after the above intervention is completed, the above mice are anesthetized, and the colon, spleen, lymph nodes, feces and serum tissues of the mice are collected for blood index detection and colon pathology analysis to determine the inflammatory changes of the mice.

[0044] In the present invention, compared with the Control group, the mice in the DSS group showed typical IBD characteristics: slow weight gain, diarrhea, bloody stool, colon structure destruction, and significantly increased fecal and blood inflammatory indicators. After oral gavage with Blautia wexlerae, the above indicators of the DSS mice were significantly improved, and there was no significant difference between the effect of oral gavage with inactivated Blautia wexlerae and that of oral gavage with active Blautia wexlerae.

[0045] The key points of the present invention are:

[0046] Step 1: Select 3-week-old C57BL / 6J male mice.

[0047] Step 2: Use 3.0% DSS drinking water and administer a specific dose of Blautia wexlerae by gavage. The gavage dose for each mouse is 1*10^9 CFU, once a day, for 9 days.

[0048] In order to make the present invention more clearly understood, a preferred embodiment is described in detail with reference to the accompanying drawings as follows:

[0049] 1 Materials and methods

[0050] 1.1 Materials

[0051] 1.1.1 Experimental animals

[0052] Male 3-week-old wild-type C57BL / 6J mice weighing 10-12g were used. The Control group was given normal drinking water and normal diet; the DSS group was given DSS drinking water and normal diet throughout the whole process; the DSS+Blautia wexlerae group was given 3.0% DSS drinking water, normal diet throughout the whole process, and Blautia wexlerae was gavaged once a day; the DSS+inactivatedBlautia wexlerae group was given 3.0% DSS drinking water, normal diet throughout the whole process, and Blautia wexlerae was gavaged once a day after heat treatment and inactivation; the Blautia wexlerae group was given normal drinking water, normal diet throughout the whole process, and Blautia wexlerae was gavaged once a day.

[0053] 1.1.2 Experimental drugs

[0054] In the specific implementation example provided, DSS was provided by MP Biomedicals, and Blautia wexlerae was provided by Mingzhou Biotechnology. The concentration of DSS drinking water was 3.0% (100 ml water: 3 g DSS), and the intragastric dose of live and inactivated Blautia wexlerae bacteria was 1*10^9 CFU.

[0055] 1.1.3 Main reagents and instruments

[0056] Cytokine and chemokine detection kit EPX260-26088-901 (Thermo Fisher Scientific), H&E dye, Luminex200.

[0057] 1.2 Experimental methods

[0058] 1.2.1 C57BL / 6J mice (n=43) were housed in an SPF environment with constant temperature (22-26°C), constant humidity (45-55%), free access to food and water, and a 12-hour day-night cycle.

[0059] 1.2.2 Randomly select 10 mice as the control group, 10 as the DSS group, 10 as the DSS+Blautia wexlerae gavage group, 8 as the DSS+inactivated Blautia wexlerae gavage group, and 5 as the DSS+Blautia wexlerae gavage group. The mice in the control group were fed with normal feed and normal drinking water. The DSS group was given 3.0% DSS drinking water and normal diet throughout the experiment; the DSS+Blautia wexlerae gavage group was given 3.0% DSS drinking water and normal diet throughout the experiment, and Blautia wexlerae was gavaged once a day at a dose of 1*10^9CFU; the DSS+inactivatedBlautia wexlerae group was given 3.0% DSS drinking water and normal diet throughout the experiment, and Blautia wexlerae was gavaged once a day after heat treatment and inactivated at a dose of 1*10^9CFU; the Blautia wexlerae group was given normal drinking water and normal diet throughout the experiment, and Blautia wexlerae was gavaged once a day at a dose of 1*10^9CFU. The experimental time was 9 days. During the experiment, the mice were weighed every day, diarrhea and blood in the stool were observed, and the amount of water consumed by the mice was recorded. At the end of the experiment, feces, serum, spleen, lymph node, colon, and small intestine tissue specimens of the mice were collected.

[0060] 2 Results

[0061] 2.1 Compared with the Control group, the weight gain of mice in the DSS group was significantly limited. Oral administration of live and inactivated Blautia wexlerae bacteria improved the weight gain ( Figure 1 A. Figure 1 B); The diarrhea and bloody stools of mice in the DSS group were significantly more severe than those in the control group. Oral administration of live and inactivated Blautia wexlerae bacteria could significantly improve DSS-induced diarrhea and bloody stools ( Figure 1 C); The spleen / body weight ratio and colon weight / colon length ratio of mice in the DSS group were significantly higher than those in the control group, and the colon length was significantly lower than that in the control group. Oral administration of live and inactivated Blautia wexlerae bacteria significantly reduced the spleen / body weight ratio ( Figure 1 D) Improve colon length ( Figure 1 E) Reduce colon edema ( Figure 1 F), the above indicators of the Blautia wexlerae group showed no significant changes compared with the control group ( Figure 1 ).

[0062] 2.2 The colon of mice in the control group had normal morphology, light yellow color, soft texture and normal length; the colon of mice in the DSS group had abnormal morphology, dark red color, congestion and edema, and significantly shortened length; after oral administration of live and inactivated Blautia wexlerae bacteria, the colon morphology of mice was significantly improved, the color was light yellow, and the length was significantly longer than that of the DSS group ( Figure 2 A). Colon H&E staining showed that the intestinal mucosal layer structure of the control group mice was intact, the goblet cells were evenly distributed, the crypts were not damaged, and there was no inflammatory cell infiltration; the colon mucosal structure of the DSS group mice was damaged, the crypts disappeared, the goblet cells were significantly reduced, and a large number of inflammatory cells infiltrated; after oral administration of live and inactivated Blautia wexlerae bacteria, the colon mucosal structure remained intact, the crypts were intact, the goblet cells were not significantly reduced, and there was no obvious inflammatory cell infiltration ( Figure 2 B).

[0063] 2.3 Calprotectin in feces of mice in the DSS group ( Figure 3 A), C-reactive protein ( Figure 3 B) serum IL-2 ( Figure 3 D) IL-6 ( Figure 3 E), IL-9 ( Figure 3 F), IL-18 ( Figure 3 I), IL-22 ( Figure 3 J), IL-23 ( Figure 3 K)、TNF-alpha( Figure 3L) levels were significantly higher than those in the Control group, and the above indicators were significantly decreased after oral administration of Blautia wexlerae. Figure 3 C) IL-13 ( Figure 3 G) showed an upward trend in the DSS group, and Blautia wexlerae decreased significantly after oral administration. IL-17A ( Figure 3 H) increased significantly in the DSS group, and Blautia wexlerae showed a significant downward trend after oral administration.

[0064] 2.4 Data Statistical Analysis

[0065] The data in the above tables are presented as Mean ± SEM and analyzed using GraphPad Prism 9 statistical software. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0066] 3 Conclusion

[0067] Both active and inactivated Blautia wexlerae can improve colonic inflammation in DSS-induced colitis mice and reduce serum inflammatory responses in mice.

[0068] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Use of probiotics Blautia wexlerae in the preparation of medicines or foods for improving or treating inflammatory bowel disease in children.

2. The use according to claim 1, characterized in that: The strain number of the probiotic Blautia wexlerae is DSM19850.

3. The use according to claim 1 or 2, characterized in that: The medicine or food uses the probiotics Blautiawexlerae as an effective component and is prepared into a dosage form acceptable in medicine or food.

4. The use according to claim 3, characterized in that: The dosage forms include powder, tablet, granule, capsule or drink.

5. The use according to any one of claims 1 to 4, characterized in that: The probiotic Blautia wexlerae is an inflammatory factor inhibitor.

6. The use according to any one of claims 1 to 5, characterized in that: The probiotic Blautia wexlerae is inactivated by heat treatment.

7. The use according to any one of claims 1 to 5, characterized in that: The probiotics Blautia wexlerae are active bacteria.

8. A drug for improving or treating inflammatory bowel disease in children, characterized in that: The medicine comprises an effective amount of probiotics Blautia wexlerae and medically acceptable auxiliary materials.

9. The drug for improving or treating inflammatory bowel disease in children according to claim 8, characterized in that: The effective dosage of the probiotic Blautia wexlerae is 1*10∧9 CFU / day.

10. A food for improving or treating inflammatory bowel disease in children, characterized in that: The food comprises an effective amount of probiotics Blautia wexlerae and auxiliary materials acceptable to food.

11. The food for improving or treating inflammatory bowel disease in children according to claim 10, characterized in that: The effective dosage of the probiotic Blautia wexlerae is 1*10∧9 CFU / day.

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