A probiotic preparation for improving intestinal mucosal barrier injury and application thereof

Abstract

The application discloses Blautia bacteria Blautia coccoides and an effective component thereof in improving intestinal function and preventing and treating inflammatory bowel disease; the Blautia bacteria belong to a new generation of probiotics (NGPs) and are derived from a healthy human intestinal microecological original flora; the Blautia bacteria can to a certain extent inhibit a TLR4 / NF-κB inflammatory signal pathway, especially specifically block phosphorylation of NF-κB p65, reduce intestinal mucosal inflammatory reactions, promote expression of tight junction proteins (ZO-1, Occludin and Claudin-1), promote recovery of intestinal epithelial barrier integrity, thereby helping to restore normal intestinal permeability, reduce mucosal inflammatory factor levels and relieve colitis and other gastrointestinal dysfunction related discomforts, and provide a safer, long-term applicable and more acceptable intestinal barrier damage improvement targeted strain scheme.

Description

Technical Field

[0001] This invention relates to the field of microecological preparations and drug application technology, specifically to Brontë cocci (… Blautia coccoides Its application in the preparation of products for improving intestinal function, repairing the intestinal physical barrier, and treating inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) represents a group of chronic, relapsing, and nonspecific inflammatory gastrointestinal diseases (including colitis and Crohn's disease) with unclear etiologies and pathogenesis. Its global incidence and prevalence are on the rise, severely impacting patients' quality of life. Modern research confirms that gut microbiota dysbiosis and impaired intestinal mucosal barrier function are important pathological bases for this type of gastrointestinal dysfunction. The intestinal epithelial barrier is a crucial interface between the host and the luminal environment; its integrity is maintained by tight junction (TJ) proteins such as ZO-1, Occludin, and Claudin-1. In IBD, the "leaky gut" phenomenon allows luminal antigens and bacteria to enter the mucosa, triggering persistent and excessive inflammation.

[0003] Traditional theories and modern medicine typically employ immunosuppressants, sulfasalazine, or traditional probiotics for intervention. In recent years, metagenomic sequencing has highlighted the importance of next-generation probiotics (NGPs) in maintaining gut health. Among them, *Brutella* (… Blautia Tight junction proteins (TJs) are among the most abundant protozoan groups in the healthy human gut. Clinically, it has been found that IBD patients not only have a severe deficiency of tight junction proteins, but also... Blautia The abundance of *Brutococcus* was significantly depleted. However, existing studies lack direct causal evidence to elucidate how specific *Brutococcus* strains protect the intestinal barrier under inflammatory conditions; the targets and molecular pathways remain unclear. Therefore, this article proposes a specific strain, *Brutococcus* (… Blautia coccoides )Specific targeted applications of DSMZ 935 in the reconstruction of gastrointestinal barrier function and improvement of inflammatory bowel disease. Summary of the Invention

[0004] The purpose of this invention is to provide a Brontë coccus ( Blautia coccoides Targeted application of this technology in improving gastrointestinal structure and function; research on the role of specific strains in combating harmful inflammatory pathways and rebuilding the mucosal barrier; and providing a basis for... Blautia coccoides This provides solid scientific evidence for clinical intervention in ulcerative colitis, leaky intestine, and physical damage to the intestinal epithelium.

[0005] To achieve the above-mentioned technical effects, the present invention is implemented through the following technical solution: Brontë cocci ( Blautia coccoides Applications of this technology in improving gastrointestinal function and repairing inflammatory diseases.

[0006] Furthermore, the aforementioned Blautia coccoides Application in ulcerative colitis and Crohn's disease.

[0007] Furthermore, the aforementioned Blautia coccoides Application in the repair of tight junction cell damage in intestinal epithelium.

[0008] Furthermore, the aforementioned Blautia coccoides The specific strain used was DSMZ 935, which was collected during the logarithmic phase of anaerobic liquid culture.

[0009] Furthermore, the aforementioned Blautia coccoides By specifically inhibiting the TLR4 / NF-κB signaling axis in intestinal tissue, suppressing the phosphorylation of NF-κB p65, downregulating pro-inflammatory markers, reversing damage to cell tight junction proteins such as ZO-1 and Occludin caused by harmful pathogens, promoting the restoration of intestinal epithelial barrier integrity, and alleviating gastrointestinal inflammatory damage.

[0010] The beneficial effects of this invention are:

[0011] 1. The core of this invention, Brouttus cocci (DSMZ 935), belongs to a new generation of preferred probiotics. When an inflammatory response occurs in intestinal tissue, it can regulate the intestinal micromucosal receptor signal and inhibit the NF-κB p65 target at the root pathway, thereby playing a powerful role in blocking inflammation and physically suturing the gaps between intestinal villi cells (epithelial integrity repair) and maintaining intestinal microecological homeostasis.

[0012] 2. Animal experiments have shown that the specific probiotic group of the present invention can effectively reduce the pathological damage (reduce disease rating) in mice with DSS-induced ulcerative colitis, block intestinal leakage in vivo (reduce blood fluorescence concentration), and reduce colonic atrophy.

[0013] 3. Cellular and clinical correlation comparisons demonstrate that the composition of the present invention can significantly restore the continuity of the monolayer intestinal epithelium severely damaged by LPS. Attached Figure Description

[0014] Figure 1 A schematic diagram comparing TJ protein and pro-inflammatory factor genes in the colonic mucosa tissue of a clinical Crohn's disease patient.

[0015] Figure 2 This is a schematic diagram illustrating the repair effect of Brontë cocci on the tight junction protein damage in Caco-2 cells according to the present invention;

[0016] Figure 3 This is a schematic diagram illustrating how Broutococcus aureus alleviates LPS-induced tight junction dysfunction by inhibiting the NF-κB p65 pathway, as described in this invention.

[0017] Figure 4This invention relates to the improvement of DSS-induced colitis in mice by Brontë cocci. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are merely examples to verify the present invention, and their implementation and application do not constitute a complete limitation.

[0019] Example 1

[0020] This embodiment provides *Bruttella* (… Blautia coccoides The practical application of this in the study of intestinal epithelial cell injury and inflammatory pathway mechanisms is as follows:

[0021] 1. Cell culture, induction and intervention

[0022] Human colorectal adenocarcinoma cells (Caco-2) were placed in culture medium supplemented with 10% FBS and cultured adherently in a cell culture incubator at 37°C and 5% CO2. The experimental groups were: normal control group (Control), model damage group (LPS group), and the *Brutococcus bradycarpa* treatment group of this invention (LPS +). Blautia coccoides The control group consisted of two groups: the inhibitor control group (LPS+TPCA-1) and the control group consisting of two groups.

[0023] The model injury group and the formulation of this invention were used to establish an intestinal cell immune inflammatory injury model. Modeling method: Cells were treated with 1 μg / mL lipopolysaccharide (LPS) for 24 h. The Brontë cocci intervention group was treated with 1×10⁻⁶ LPS 6 h before modeling. 9 The bacterial suspension was pretreated with CFU / mL cultured bacteria; the pathway validation group was treated alone with the NF-κB specific inhibitor TPCA-1 for comparison.

[0024] 2. Western Blot Analysis

[0025] Total protein was extracted from cells and its concentration was determined. After electrophoresis and electroporation, the protein was transferred to a PVDF membrane and blocked with 5% BSA. The membrane was incubated overnight at 4°C with primary antibodies against the target proteins, including ZO-1, Occludin, Claudin-1, TLR4, NF-κB p65, and p-p65. Changes in protein expression were observed using ECL chemiluminescence.

[0026] 3. Immunofluorescence assay (IF)

[0027] After preparing cell slides, fixation and blocking were performed, followed by incubation overnight at 4°C with primary antibodies against the target proteins, including ZO-1, Occludin, and Claudin-1. Changes in protein distribution were observed using fluorescence microscopy.

[0028] 4. Experimental Results and Conclusions

[0029] Compared to the Control group, the LPS group showed a significant decrease in the protein and gene transcriptional expression levels of ZO-1, Occludin, and Claudin-1 in intestinal epithelial cells, and exhibited obvious gap pores and punctate disruptions under immunofluorescence microscopy. In contrast, the Brontë cocci group of this invention maintained a firm and continuous "honeycomb" interconnected network structure even after LPS disruption. Mechanistic studies revealed that the LPS group induced intense activation of TLR4 / NF-κB and hyperphosphorylation of p65; the addition of the probiotics of this invention achieved the same function as the specific blocking drug (TPCA-1) in preserving the receptor cell barrier structure.

[0030] Example 2

[0031] This embodiment provides *Bruttella* (… Blautia coccoides The actual preclinical translational application in mice with ulcerative colitis follows these steps:

[0032] 1. Modeling, grouping, and drug administration

[0033] Five-week-old healthy male C57BL / 6 mice were randomly divided into a control group, a model group (DSS group), and an experimental validation group (DSS+ group) after adaptive feeding. Blautia coccoides Group).

[0034] A. The control group was given a normal diet, while the model group and the experimental group were treated with 3% sodium dextran sulfate (DSS) added to their drinking water for 7 consecutive days to induce acute modeling.

[0035] B. Before and during the challenge and modeling period, the experimental verification group received 10 ml / kg of the Brontë cocci solution of this invention orally via gavage daily. 9 (CFU / mL), the model group was simultaneously gavaged with the same weight of physiological saline.

[0036] 2. Intestinal leakage and physiological index detection

[0037] Throughout the experiment, the mice were examined for changes in body weight, fecal matter, and occult blood to establish a disease activity index for DAI. On the last day, the mice were fasted and ingested a fixed amount of fluorescein isothiocyanate-dextran (FITC-Dextran 4kDa). The physical leakage rate of the intestinal epithelial reticulum was determined by measuring the fluorescence value of serum leaked into the external circulation in the model group and the drug-treated group after the experiment was interrupted. Subsequently, intestinal segments were harvested, measured, and H&E tissue sections were performed for observation.

[0038] 3. Results and Conclusions

[0039] Physical examination results showed that DSS in the model group caused significant pathogenicity (weight loss and increased DAI score) from day four onwards, with severe colonic atrophy. In contrast, the Brontë cocci intervention group treated with this invention maintained strong and stable body weight, and the colon length showed resilience comparable to the healthy group. More importantly, in the assessment of intestinal permeability (leakage), DSS caused a surge in serum fluorescein in mice (due to barrier collapse and permeation), while the application of this invention resulted in a statistically significant decrease in serum fluorescein titer.

[0040] The above results confirm that the Brontë cocci selected in this invention effectively improves the physiological fulminant of ulcerative colitis in mice, alleviates intestinal leakage and secondary immunity caused by increased mucosal permeability, and has great market potential and medical auxiliary capabilities for the repair of human gastrointestinal inflammation and digestive dysfunction.

Claims

1. Brontë cocci ( Blautia coccoides Its application in the preparation of products for the treatment of inflammatory bowel disease.

2. The application according to claim 1, characterized in that, The Brontë cocci ( Blautia coccoides Application of ) in ulcerative colitis and Crohn's disease.

3. The application according to claim 1, characterized in that, The Brontë cocci ( Blautia coccoides Application of ) in intestinal epithelial cell and physical barrier damage.

4. The application according to claim 1, characterized in that, The Brontë cocci ( Blautia coccoides The specific strain is strain DSMZ 935 from the resource library.

5. The application according to claim 1, characterized in that, The concentration of the preparation containing *Brutococcus bradycarpa* used in the preparation of products for improving gastrointestinal function is not less than 1 × 10⁻⁶. 9 CFU / mL live bacterial preparations, bacterial cell enrichments, or lyophilized powders.

6. The application according to claim 1, characterized in that, The Broutococcus species improves the intestinal microenvironment, inhibits abnormal activation of the TLR4 / NF-κB pathway and phosphorylation of NF-κB p65, reduces intestinal mucosal inflammation, upregulates the expression of tight junction proteins in intestinal epithelium, and promotes the restoration of intestinal epithelial barrier integrity, thereby helping to restore normal intestinal permeability and alleviate gastrointestinal inflammation and related discomfort.

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