Compound probiotics for preventing and treating acute intestinal inflammation in mice and application thereof

Abstract

The application belongs to the technical field of biological agents, and discloses a compound probiotic for preventing and treating acute intestinal inflammation of mice and application. The compound probiotic comprises: Kluyveromyces marxianus C2 with a preservation number of CCTCC NO: M 2014059, Bacillus subtilis KC with a preservation number of CCTCC NO: M 20211444, and Lactobacillus acidophilus. The applicant finds that the compound probiotic preparation can significantly relieve the clinical symptoms and the pathological damage of the colon tissue caused by DSS, significantly reduce the DAI score, and significantly improve the intestinal barrier function by constructing an IBD animal model and treating the mice by gavage with the compound probiotic.

Description

Technical Field

[0001] This invention belongs to the field of biological agent technology and relates to a compound probiotic for the prevention and treatment of acute intestinal inflammation in mice and its application. Background Technology

[0002] Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a chronic, nonspecific inflammatory bowel disease of unknown origin. Clinical symptoms include diarrhea, bleeding, abdominal pain, fever, and weight loss. The different clinical symptoms of patients depend mainly on the type, location, and severity of the disease. Its cause is closely related to gut microbiota dysbiosis. Changes in the composition or diversity of gut microbiota can lead to gut microbiota dysbiosis, affecting normal intestinal metabolic processes, disrupting the homeostasis of the intestinal immune system, and promoting or inducing the occurrence and development of the disease.

[0003] Currently, the main types of drugs used clinically to treat IBD include three categories: aminosalicylic acids, glucocorticoids, and immunosuppressants. Long-term use of these drugs can lead to symptoms such as weakened immunity, and IBD relapse can occur after discontinuation. Furthermore, these drugs only provide symptomatic treatment and cannot cure IBD. Therefore, there is an urgent need to find a safe and effective drug to treat IBD.

[0004] Studies have shown that gut microbiota plays a crucial role in the pathogenesis of inflammatory bowel disease (IBD). Reshaping the gut microbiota, especially by supplementing with functional strains that can repair disordered microbiota, has outstanding potential in alleviating IBD and may become a new tool for the prevention and treatment of acute or chronic diarrhea. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a compound probiotic, which includes Kluyveromyces martensii C2, with accession number CCTCC NO: M 2014059; Bacillus subtilis KC, with accession number CCTCC NO: M 20211444; and Lactobacillus acidophilus.

[0006] Another objective of this invention is the application of the above-mentioned compound probiotics in the preparation of a drug to relieve symptoms of inflammatory bowel disease in mice.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] This invention screened out three probiotic strains, which, when used as a compound preparation, constitute a compound probiotic that can prevent and treat IBD in mice and has a synergistic effect.

[0009] The scope of protection of this invention includes:

[0010] A compound probiotic for preventing and treating acute intestinal inflammation in mice, the compound probiotic comprising: Kluyveromyces martensii C2, preservation number CCTCC NO: M 2014059 (CN103981129A); Bacillus subtilis KC (CN114231460A), preservation number CCTCC NO: M 20211444; and Lactobacillus acidophilus.

[0011] The Lactobacillus acidophilus in the compound probiotics mentioned above is sourced from commercial channels.

[0012] Preferably, the ratio of the effective bacterial concentrations of the compound probiotics described above, namely Kluyveromyces martensii C2, Bacillus subtilis KC, and Lactobacillus acidophilus, is 1~3:1~3:1~3.

[0013] In the above-mentioned compound preparation, preferably, the effective bacterial concentration ratio of Kluyveromyces martensii C2, Bacillus subtilis KC and Lactobacillus acidophilus is 1:1:1.

[0014] Preferably, in the compound preparations described above, the effective bacterial concentration of the compound microbial agent is 0.1~9.0×10⁻⁶. 9 CFU / g.

[0015] Preferably, in the compound preparations described above, the effective bacterial concentration of the compound microbial agent is 1.0~9.0×10⁻⁶. 9 CFU / g.

[0016] The above-mentioned compound microbial agent is used in the preparation of drugs for the treatment or prevention of inflammatory bowel disease.

[0017] In the above-described applications, preferably, the drug includes excipients, which are pharmaceutical or food-acceptable excipients.

[0018] In the above-described applications, preferably, the dosage form of the compound microbial agent includes powder, capsules, or tablets.

[0019] The inflammatory bowel disease described above refers to acute colitis.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. This invention provides a compound probiotic containing Kluyveromyces martensii, Bacillus subtilis and Lactobacillus acidophilus. Experiments have shown that this compound probiotic preparation can significantly alleviate clinical symptoms and colonic tissue pathological damage caused by DSS, significantly reduce DAI score, and significantly improve intestinal barrier function.

[0022] 2. The present invention provides a strain of Kluyveromyces rubrum that has the advantages of simple production process and low price. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the weight monitoring results and DAI score change trends provided in Embodiment 2 of the present invention;

[0024] Where: A represents the trend of mouse body weight change, and B represents the trend of mouse DAI score change;

[0025] Note: * indicates significance compared to the control group: P < 0.05; *** indicates significance: P < 0.001; # indicates significance compared to the DSS group: P < 0.05; ### indicates significance: P < 0.001.

[0026] Figure 2 The image and length statistics diagram of the colon tissue provided in Embodiment 2 of the present invention.

[0027] Figure 3 This is a schematic diagram of the morphological structure of colon tissue provided in Embodiment 2 of the present invention.

[0028] Figure 4 This is a schematic diagram of the pathological changes of the spleen provided in Embodiment 2 of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to embodiments. It should be understood that the embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0030] Unless otherwise specified, the experimental methods used in the examples described below are conventional methods. Reagents or instruments used in the examples described below, unless otherwise specified, are commercially available products.

[0031] In this invention, Kluyveromyces martensii C2, with accession number CCTCC NO: M2014059 and publication number CN103981129A; Bacillus subtilis KC, with accession number CCTCC NO: M20211444 and publication number CN114231460A; and Lactobacillus acidophilus were purchased from Beijing Haoshiwo Biotechnology Co., Ltd.

[0032] Example 1:

[0033] Preparation of the compound probiotic preparation and other control groups provided by this invention:

[0034] In this embodiment, the fermentation broths of the three strains were all treated with spray drying, and the effective bacterial concentration after spray drying was 1.0 × 10⁻⁶. 9 CFU / g. In this example, the effective bacterial concentration of the total bacteria in the compound microbial agent is 1.0 × 10⁻⁶. 9 CFU / g.

[0035] Compound probiotic group:

[0036] Kluyveromyces maculae C2, Bacillus subtilis KC, and Lactobacillus acidophilus were mixed in a mass ratio of 1:1:1.

[0037] Comparative Example 1:

[0038] Kluyveromyces maculae C2 and Bacillus subtilis KC were mixed at a mass ratio of 1:1.

[0039] Comparative Example 2:

[0040] Kluyveromyces maculae C2 and Lactobacillus acidophilus were mixed at a mass ratio of 1:1.

[0041] Comparative Example 3:

[0042] Kluwer yeast C2.

[0043] Comparative Example 4:

[0044] Bacillus subtilis KC.

[0045] Comparative Example 5:

[0046] Lactobacillus acidophilus.

[0047] Example 2:

[0048] Verification test on the efficacy of compound probiotics in the prevention and treatment of inflammatory bowel disease:

[0049] 1.1 Experimental Animals

[0050] Eighty SPF-grade male C57BL / 6 mice (6-8 weeks old, 20g±2g).

[0051] 1.2 Test methods and judgment criteria

[0052] Eighty male C57BL / 6 mice were randomly divided into eight groups, as shown in Table 1. After a 7-day pre-feeding period, the mice were administered probiotics via gavage according to Table 1 for two consecutive weeks. On the 8th day after probiotic administration, the mice had free access to a 3% sodium dextran sulfate (DSS) aqueous solution for 7 consecutive days. All bacterial strains used in the experiment were preserved by Wuhan Keqian Biotechnology Co., Ltd. The blank control group received no gavage or modeling treatment.

[0053] Table 1 Experimental Groups and Schemes

[0054]

[0055] During the trial, according to Figure 1 (Day 1 was defined as the start of the experiment, when the drinking water was changed to a 3% DSS aqueous solution, and Day 8 was the end of the experiment.) Mouse weight was recorded daily, stool characteristics were monitored, and occult blood tests were performed to calculate the Disease Activity Index (DAI). The DAI was determined by weight loss, stool consistency, and fecal occult blood. Weight loss scores were as follows: 0 points, <1%; 1 point, 1%-5%; 2 points, 5%-10%; 3 points, 10%-20%; 4 points, >20%. Stool characteristics were categorized into three levels: 0 points, normal stool; 2 points, loose stool; 4 points, watery stool. Fecal occult blood was scored using the following scale: 0 points, negative; 2 points, positive; 4 points, obvious bloodstains in the stool.

[0056] Mice were sacrificed on day 7, and the colon was quickly removed from the mice. Its length was measured and photographed. Part of the colon was immersed in 4% paraformaldehyde overnight for paraffin section preparation. HE and AB-PAS staining were then performed to observe the inflammation and damage in the colon. The histological scoring of the colon of IBD mice was performed according to the following criteria: (1) Severity of inflammation: 0 points: none; 1 point: mild; 2 points: moderate; 3 points: severe. (2) Spread of inflammation: 0 points: none; 1 point: mucosa; 2 points: mucosa and submucosa; 3 points: transmural. (3) Recess damage: 0 points: none; 1 point: basal 1 / 3 damaged; 2 points: basal 2 / 3 damaged; 3 points: only the surface epithelium is incomplete; 4 points: overall crypt and epithelium are lost. (4) Percentage of involvement: 1 point: 1~25%; 2 points: 26~50%; 3 points: 51~75%; 4 points: 76~100%. Multiply the score for each indicator by the percentage of affected data for each cross-section, and then sum the calculated scores for the three criteria.

[0057] Organ index: Mice in each group were euthanized. The heart, liver, spleen (partially immersed in 4% paraformaldehyde to prepare HE), lungs and kidneys of each mouse were removed and weighed. Excess tissue and bloodstains were removed before weighing.

[0058] Organ index = immune organ mass (mg) / mouse body mass (g).

[0059] RT-qPCR: RNA was extracted from a portion of the colon, and the gene expression of inflammatory factors TNF-α, IL-1β, anti-inflammatory factor IL-10, and intestinal mucosal barrier factors ZO-1, Cludin-5, and Occludin was detected by RT-qPCR.

[0060] 1.3 Test Results

[0061] 1.3.1 Prevention and Treatment of Clinical Symptoms of DSS-Induced Inflammatory Bowel Disease in Mice by Compound Probiotics

[0062] During the experiment, clinical observation revealed that the NC group mice had bright black-gray fur, were agile and active, had normal appetites, and showed no diarrhea or bloody stools. Mice drinking water supplemented with 3% DSS experienced varying degrees of weight loss, accompanied by lethargy and reduced food intake, with later symptoms including bloody stools and diarrhea. Mice in the compound probiotic group and the control group showed varying degrees of improvement in their mental state, food intake, and diarrhea / bloody stools.

[0063] Table 2. Statistics on mouse fecal shape

[0064]

[0065] Table 3. Statistics of fecal occult blood in mice

[0066]

[0067] 1.3.2 Body weight and intestinal inflammation activity score in mice with DSS-induced inflammatory bowel disease treated with compound probiotics

[0068] Throughout the experiment, the body weight of mice in the NC group showed a slight upward trend. Mice in the DSS group, the probiotic groups of each example, and the comparative probiotic group experienced varying degrees of body weight loss after drinking 3% DSS aqueous solution. After 14 days of intervention with compound probiotics, BLK significantly inhibited the trend of body weight loss in IBD mice (P < 0.01). Figure 1 As shown in Figure A, mice that drank a 3% DSS aqueous solution exhibited symptoms including weight loss, lethargy, and reduced appetite, followed by diarrhea and bloody stools. The probiotic groups and comparative groups in each example alleviated these symptoms to varying degrees. The BLK group mice showed only mild diarrhea with no visible blood in their feces, and significantly reduced the DAI score in mice with acute IBD (P < 0.001). This indicates that adding BLK probiotic solution can alleviate DSS-induced inflammatory bowel disease in mice, as shown in the DAI results. Figure 1 As shown in B.

[0069] Table 4. Disease Activity Index (DAI) Scores for Mice

[0070]

[0071] Note: Different letters in the same row (column) indicate significant differences (P<0.05); the same letter or no letter in the shoulder indicates no significant differences (P>0.05). The same applies below.

[0072] 1.3.5 Prevention and Treatment of Colonic Changes in DSS-Induced Inflammatory Bowel Disease Mice by Compound Probiotics

[0073] Changes in colon length in mice are an important indicator for assessing the severity of IBD. Colon length statistics showed that, compared to the control group, mice treated with DSS had significantly shorter colon lengths (P<0.001), indicating successful model induction. However, treatment with six compound probiotics significantly increased colon length in the BK, LK, and BLK groups (P<0.05), with BLK probiotics showing a more pronounced preventative effect (P<0.001). Figure 2 As shown.

[0074] Table 5. Statistics on colon length in mice

[0075]

[0076] 1.3.6 Pathological Changes in Colonic Tissue of Mice with DSS-Induced Inflammatory Bowel Disease Treated by Compound Probiotics

[0077] To further evaluate the alleviating effect of probiotics in each group on IBD in mice, HE staining and AB-PAS staining were performed on mouse colon tissue. It was observed that the colon tissue structure of the NC control group mice was clear and intact, with a large number of goblet cells, and the intestinal mucosa structure was complete, without inflammatory cell infiltration. In the DSS model group mice, the intestinal mucosa structure was significantly damaged, with tissue edema, reduced or absent glands and goblet cells, and a large number of inflammatory cells infiltrating the mucosa, submucosa, and muscularis propria. The colonic mucosa structure of the intervention groups was significantly closer to normal than that of the model group, but still showed signs of glandular damage, reduced goblet cells, and a small amount of inflammatory cell infiltration. Figure 3 As shown.

[0078] 1.3.7 Pathological scoring of colonic tissue in mice with DSS-induced inflammatory bowel disease treated with compound probiotics

[0079] HE and AB-PAS staining were used to determine the pathological condition of colonic tissue in different groups of mice. The results are shown in Table 3. Except for the NC control group, the other 7 groups showed an increase in colonic tissue pathological scores under DSS induction. Among them, the DSS group showed a significant increase in colonic tissue pathological scores (P < 0.001). After intervention with the compound probiotic BLK, the above symptoms in the colonic tissue of IBD-score mice were improved to a certain extent, the histopathological scores were significantly reduced (P < 0.05), and the number of goblet cells increased (P < 0.05). The results indicate that the BLK group bacterial solution can effectively reduce the pathological changes in mouse colonic tissue caused by inflammatory bowel disease. Compared with other probiotic groups, the three probiotics have a significant synergistic effect.

[0080] Table 6. Morphological and pathological scores of mouse colon tissue; statistics on the number of goblet cells in colon tissue.

[0081]

[0082] 1.3.8 Compound probiotics in preventing organ changes in mice with DSS-induced inflammatory bowel disease

[0083] Compared with the NC control group, the spleen index of mice in the DSS model group was significantly increased (P<0.001), while the indices of the heart, liver, lungs, and kidneys showed no significant changes (P>0.05). Compared with the DSS model group, the BLK probiotic solution significantly reduced the spleen index (P<0.001), while the spleen index of other comparative probiotic groups showed no significant changes (P>0.05), indicating that the addition of the compound probiotic BLK had a mitigating effect on inflammation induced by IBD.

[0084] Table 7. Mouse organ index (%)

[0085]

[0086] 1.3.9 Pathological Changes in the Spleen of Mice with DSS-Induced Inflammatory Bowel Disease Treated by Compound Probiotics

[0087] In the NC control group, the spleen tissue of mice showed normal morphology, dense structure, good white pulp morphology, round or oval splenic bodies, obvious germinal centers, and a reasonable ratio and clear boundary between white and red pulp. In the DSS model group, the overall spleen tissue structure was abnormal, with indistinct boundaries between red and white pulp, significant atrophy of splenic nodules, increased red pulp, sparse lymphocyte arrangement, no obvious lymphocyte necrosis, and extensive infiltration of multinucleated giant cells. Compared with the DSS model group, the pathological morphology of spleen tissue in all intervention groups was improved, damage to the white pulp area was reduced, the boundary between white and red pulp was clearer, and the structure tended to be normal. Figure 4 As shown.

[0088] 1.3.8 Changes in colonic inflammatory factors in mice with DSS-induced inflammatory bowel disease treated with compound probiotics

[0089] Compared with the NC control group, the DSS model group showed a significant decrease in the level of the anti-inflammatory factor IL-10 in the colon tissue (P<0.001), and a significant increase in the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 (P<0.001). Compared with the DSS model group, the BS, LA, KM, BK, LK, and BLK groups showed a significant increase in the expression level of the anti-inflammatory factor IL-10 (P<0.05), and a significant decrease in the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 (P<0.05). The BLK group showed the best effect of intervention with the compound bacterial solution compared to the other five groups, exhibiting a significant synergistic effect. It also demonstrated a certain protective and repairing effect on the intestinal immune barrier.

[0090] Table 8. Changes in inflammatory factors in mouse colon tissue.

[0091]

[0092] 1.3.9 Changes in intestinal barrier factors in the colon of mice with DSS-induced inflammatory bowel disease treated with compound probiotics

[0093] Tight junction proteins, composed of transmembrane proteins (such as Claudin-1 and Occludin) and cytoplasmic proteins (ZO-1), are crucial for maintaining the integrity of the intestinal mucosal barrier. Compared with the NC control group, the DSS model group significantly reduced the expression levels of ZO-1, Occluding, and Claudin-1 in the mouse colon (P<0.001); the BS, KM, BK, LK, and BLK groups all significantly increased the expression levels of ZO-1, Occluding, and Claudin-1 (P<0.05). This indicates that the addition of compound probiotics and comparative probiotics can improve the permeability of the intestinal mucosal barrier in mice, which is beneficial for alleviating the damage to the colonic structure caused by DSS infection. Compared with other probiotic groups, the three probiotics have a significant synergistic effect.

[0094] Table 9 Changes in colonic intestinal barrier factors

[0095]

[0096] The above results indicate that the compound probiotic preparation provided by the present invention can significantly alleviate the clinical symptoms and colonic tissue pathological damage caused by DSS, significantly reduce the DAI score, and significantly improve intestinal barrier function, and has great application potential.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound probiotic for preventing and treating acute intestinal inflammation in mice, wherein the compound probiotic comprises: Including Kluwer yeast ( kluyeromyces marxianus C2, preservation number CCTCC NO: M 2014059; Bacillus subtilis ( Bacillus subtilis KC, with accession number CCTCC NO: M 20211444 and Lactobacillus acidophilus ( Lactobacillus acidophilus ).

2. The compound probiotic according to claim 1, characterized in that: The effective bacterial concentration ratio of Kluyveromyces maculae C2, Bacillus subtilis KC and Lactobacillus acidophilus is 1~3:1~3:1~3.

3. The compound probiotic according to claim 1, characterized in that: The effective bacterial concentration ratio of Kluyveromyces maculae C2, Bacillus subtilis KC and Lactobacillus acidophilus is 1:1:

1.

4. The compound probiotic according to claim 2, characterized in that: The effective bacterial concentration of the compound probiotics is 0.1~9.0×10⁻⁶. 9 CFU / g.

5. The compound probiotic according to claim 3, characterized in that: The effective bacterial concentration of the compound probiotics is 1.0~9.0×10⁻⁶. 9 CFU / g.

6. The use of the compound probiotics according to claim 1 in the preparation of drugs for treating or preventing inflammatory bowel disease.

7. The application according to claim 6, wherein the drug includes excipients, which are pharmaceutical or food-acceptable excipients.

8. The application according to claim 7, wherein the dosage form of the drug includes powder, capsules or tablets.

9. The application according to claim 6, wherein the inflammatory bowel disease is acute colitis.

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