Use of roseburia hominis in the preparation of a product for the prevention and treatment of inflammatory bowel disease

Abstract

The application discloses Roseburia hominis The application discloses an application in preparation of a product for preventing and treating inflammatory bowel disease, and belongs to the technical field of biological medicine. R. hominis The application proves that, R. hominis The surface protein SBP of the application can significantly reduce the colitis induced by dextran sulfate sodium, reduce the disease activity index, relieve the colon shortening, reduce the tissue inflammatory damage, and improve the intestinal mucosal barrier function. It is also found that the intestinal epithelial fucosylation level is a key host factor affecting the colonization and curative effect. R. hominis Only when the level of the subject is not significantly reduced, R. hominis The application can effectively colonize and play the anti-inflammatory and barrier protection roles. Moreover, the application can increase the anti-inflammatory and barrier protection curative effect of a TNF-alpha inhibitor. The application provides a personalized treatment strategy based on the host fucosylation state for inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to... Human rosebuds The application of its surface sugar ABC transporter substrate-binding protein SBP in the prevention, treatment or adjuvant therapy of intestinal epithelial fucosylation levels in patients with inflammatory bowel disease who did not show a significant decrease. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease of unknown etiology, characterized by diarrhea, bloody and mucous stools, and abdominal pain as its main symptoms. Its global incidence and prevalence have been steadily increasing over the past thirty years, and its chronic and recurrent course imposes a significant disease burden. The pathogenesis of UC is generally considered to be closely related to genetic susceptibility, immune system abnormalities, environmental factors, and gut microbiota dysbiosis.

[0003] Current treatments for ulcerative colitis (UC) primarily include aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics. While these treatments can alleviate inflammatory activity to some extent, they still present challenges such as limited efficacy in some patients, significant individual variability, difficulty in long-term maintenance, and a high relapse rate after discontinuation. Anti-TNF biologics also have limitations, including primary non-response and secondary loss of response; therefore, new treatment strategies need to be explored.

[0004] Gut microbiota dysbiosis is a key characteristic of ulcerative colitis (UC). UC patients exhibit reduced gut microbiota diversity, with a composition significantly different from healthy individuals. This is manifested in an increased abundance of potentially key pathogenic bacteria in UC and a decrease in various commensal bacteria associated with gut homeostasis. Dysbiosis can lead to impaired intestinal mucosal barrier function, increased intestinal permeability, reduced protective metabolites, and the induction of an immune-inflammatory cascade, thus promoting the development and progression of UC. Therefore, gut microbiota intervention shows potential in the adjunctive treatment of UC. Fecal microbiota transplantation (FMT) and probiotic / next-generation probiotic therapies are considered promising for alleviating intestinal inflammation by remodeling the gut microbiota, improving the mucosal barrier, and modulating the immune response. Existing studies have shown that FMT can induce clinical or endoscopic remission in some UC patients; however, efficacy varies considerably between studies, and overall reproducibility and stability remain limited. This may be because simply supplementing with exogenous microorganisms does not guarantee stable therapeutic benefits; its efficacy may be influenced by host factors, strain colonization ability, and the host-microbiota interaction state.

[0005] Glycosylation status in the host gut microenvironment is considered a crucial factor influencing host-microbiota interactions. Fucosylation, primarily mediated by fucosyltransferase 2 (FUT2), is an important form of glycosylation for maintaining gut symbiotic homeostasis. Previous studies have shown that differences in FUT2 and intestinal epithelial fucosylation levels can lead to variations in gut microbiota composition and are associated with susceptibility to inflammatory bowel disease. Furthermore, host fucosylation status may further affect the interaction between specific bacterial strains and the host, thereby influencing bacterial recognition, adhesion, and colonization of the host surface. Therefore, host fucosylation status may be a significant host factor influencing the colonization efficiency and therapeutic efficacy of specific beneficial bacteria; however, current technologies still lack a full understanding of its role in the colonization and function of specific therapeutic strains.

[0006] butyric acid bacteria Human rosebuds It is one of the important symbiotic anaerobic bacteria in the human gut, and its abundance is significantly reduced in UC patients. Studies have shown that... R.man It may be involved in regulating intestinal barrier function, host immune response, and maintenance of intestinal homeostasis; therefore, with R.man Beneficial bacteria or their related preparations show promising research and application prospects as interventions for UC. However, current technologies for the treatment of UC still have significant limitations. First, after exogenous beneficial bacteria enter the host gut, they often fail to achieve sustained, stable, and functional colonization, leading to significant differences in therapeutic effects among different individuals. Second, existing research largely focuses on changes in bacterial abundance, disease correlation analysis, or macroscopic host background differences. The key factors influencing the colonization efficiency of specific beneficial bacteria, especially how the host microenvironment affects the colonization efficiency, survival ability, and therapeutic effects of specific strains, remain insufficiently understood, and there is a lack of technical solutions that can be directly applied to the development and optimization of bacterial agents.

[0007] Given that existing bacteria or their preparations still suffer from problems such as insufficient colonization efficiency, limited duration of efficacy, and significant individual variability in the treatment of ulcerative colitis, it is necessary to develop novel [treatments / methods]. Human rosebuds This is a fundamental therapeutic technology that enables the treatment to better adapt to the host's gut microenvironment, improve its ability to colonize and exert a sustained effect, and enhance the efficacy of other treatments. It has significant scientific and clinical translational value. Summary of the Invention

[0008] To address the aforementioned problems in the existing technology, the purpose of this invention is to provide a method based on the host FUT2 genotype. Human rosebudsApplication in the preparation of personalized treatment drugs for ulcerative colitis. This strain can sensitize the anti-inflammatory and barrier-protective effects of TNF-α inhibitors, enabling precise intervention in inflammatory bowel disease. This method is non-invasive, accurate, and suitable for clinical application and promotion.

[0009] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: This invention provides Human rosebuds Application in the preparation of products for the prevention and treatment of inflammatory bowel disease, said products comprising Human rosebuds The product is a live bacterium or its surface glyco-ABC transporter substrate-binding protein SBP, and is intended for use in subjects with inflammatory bowel disease in whom the level of intestinal epithelial fucosylation is not significantly reduced.

[0010] Furthermore, those whose intestinal epithelial fucosylation levels were not significantly reduced included subjects whose FUT2 gene was functionally wild-type.

[0011] Furthermore, the product also contains a TNF-α inhibitor.

[0012] Furthermore, the product is administered in combination with a TNF-α inhibitor.

[0013] Furthermore, the product is used to sensitize the anti-inflammatory and / or intestinal barrier protective effects of TNF-α inhibitors.

[0014] Furthermore, the TNF-α inhibitor includes an anti-TNF-α antibody.

[0015] Furthermore, the SBP protein mediates by binding to SLC3A2 on the surface of intestinal epithelial cells. Rosemary of man Intestinal colonization.

[0016] Furthermore, the inflammatory bowel disease includes ulcerative colitis.

[0017] Furthermore, the aforementioned Human rosebuds include Human rosebuds strain A2-183T.

[0018] Furthermore, the product includes pharmaceuticals.

[0019] This invention addresses the shortcomings of existing technologies for treating ulcerative colitis, such as significant individual variability, lack of clear host stratification criteria, and non-responsiveness to TNF-α inhibitors. It provides... Human rosebuds Application in the preparation of products for the prevention, treatment, or adjunctive treatment of inflammatory bowel disease. Compared with the prior art, the present invention has the following beneficial effects: 1. This invention confirms Human rosebudsSBP protein can significantly alleviate DSS-induced colitis, reduce disease activity index scores, alleviate colonic shortening, reduce tissue inflammatory damage, and improve intestinal mucosal barrier function.

[0020] 2. Discovery of this invention Human rosebuds The therapeutic effect is closely related to the level of intestinal colonization, thus clarifying the important limiting factors affecting the efficacy of bacterial agents and providing a basis for improving the application effect of bacterial agents.

[0021] 3. This invention clarifies that FUT2-mediated intestinal epithelial fucoidylation is a regulatory mechanism. Human rosebuds It is an important host factor that exerts anti-inflammatory and barrier protection effects, making up for the lack of clarity in the host-microbiota interaction mechanism in existing technologies.

[0022] 4. Discovery of this invention Human rosebuds It can enhance the therapeutic effect of TNFα inhibitors on colitis, providing a new application method for the combination therapy and sensitization therapy of ulcerative colitis.

[0023] 5. Based on the host FUT2 genotype or related fucosylation status, this invention can provide a theoretical basis and application prospects for individualized bacterial agent treatment and precise combination therapy for patients with ulcerative colitis. Attached Figure Description

[0024] Figure 1 for R. man Effects of gavage on DSS-induced acute colitis in mice. A: Schematic diagram of the experimental protocol; B: Body weight change curve; C: Disease Activity Index (DAI) score; D: Representative H&E staining images of colonic tissue; E: Histopathological score; F: Representative colon photographs; G: Colon length statistics; H: qPCR detection results of inflammatory factors and intestinal barrier-related molecules.

[0025] Figure 2 for R. man Detection of colonization levels in the gut. A: Fluorescence in situ hybridization (FISH) detection. R. of man Planting (red fluorescence); B: R. man DNA PCR detection; C: qPCR quantitative analysis R. man Abundance.

[0026] Figure 3 The effect of FUT2-mediated intestinal epithelial fucosylation on R. hominis adhesion was investigated. AB: Immunofluorescence staining was used to detect the effects of Fut2 knockdown / overexpression and UEA-I blockade on bacterial adhesion; CD: Flow cytometry was used to quantitatively analyze bacterial adhesion rate; EF: Western blot and qPCR were used to verify the effects of Fut2 knockdown / overexpression.

[0027] Figure 4 To screen for proteins that interact with intestinal epithelial cell membranes R. man Membrane proteins. A: Far-western blotting results; B: Mass spectrometry identification of candidate proteins; C: Biotin pull-down detection results; D: Mass spectrometry identification of candidate proteins.

[0028] Figure 5 To verify the interaction between SBP and SLC3A2 in intestinal epithelial cells. A: Purification of GST-SBP recombinant protein; B: GST pull-down silver staining results; C: Mass spectrometry identification of SLC3A2 as an SBP interacting protein.

[0029] Figure 6 SLC3A2-mediated R. man Functional validation of adhesion. A and B: Western blot and qPCR validation of SLC3A2 knockdown / overexpression effects; C: Immunofluorescence staining detection of the effect of SLC3A2 knockdown / overexpression on bacterial adhesion.

[0030] Figure 7 This is a schematic diagram of the grouping scheme for animal experiments.

[0031] Figure 8 for R. man Comparison of treatment effects in wild-type and Fut2 knockout mice. A: Body weight change curve; B: DAI score; CD: Colon length statistics and representative photographs; EF: H&E staining and histopathological scores.

[0032] Figure 9 for R. man Effects on the expression of intestinal tight junction proteins (immunofluorescence staining).

[0033] Figure 10 The effect of SBP protein on a TNF-α-induced intestinal epithelial cell inflammation model (qPCR detection).

[0034] Figure 11 for R. man Schematic diagram of the grouping scheme for experimental animals sensitized by combined TNF-α inhibitors.

[0035] Figure 12 for R. man The therapeutic sensitization effect of combined TNF-α inhibitors on colitis. A: Body weight change curve; B: DAI score; C: Representative colon photographs; D: Representative H&E stained colonic tissue images; E: Colon length statistics and histopathological score.

[0036] Figure 13 for R. manComparison of colonization levels in the intestines of wild-type and Fut2 knockout mice. Detailed Implementation

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0038] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0039] Example 1 I. Experimental Methods 1. R.man Bacterial culture 1) Human rosebuds The A2-183T strain (=DSM 16839T = NCIMB 14029T) was purchased from Wuhan First Light Industry Research Institute (HBCICC59004).

[0040] 2) Culture medium preparation: Add 5.25 g ATCC Medium 1490 medium, 0.5 g glucose, 0.1 g maltose, and 0.1 g soluble starch to every 100 ml of double-distilled water. Boil under N2, 10% H2, and 10% CO2 atmospheres, then cool and adjust the pH to 7.0. Aliquot 7 ml of the solution into test tubes containing meat particles (meat particle to liquid ratio 1:5) under the same atmosphere. Sterilize by steaming at 121℃ for 15 min.

[0041] 3) Human rosebuds Culture: Add an appropriate amount of bacterial solution to each culture tube and incubate overnight in an anaerobic environment at 37 °C. After about 16 hours, the bacteria will be in the logarithmic growth phase.

[0042] 2. Construction and evaluation of a mouse model of DSS-induced acute intestinal inflammation Thirty male, 8-week-old, uniformly weighted intestinal epithelium-specific Fut2 knockout (Fut2ΔIEC) mice and 30 wild-type mice with the same genetic background were purchased and randomly divided into six groups of equal size: wild-type control group (WT Con), wild-type DSS group (WT DSS), wild-type control group, ... R.man Gavage group (WT DSS+Rh), Fut2 ΔIEC Mouse control group (Fut2) Δ IEC Con), Fut2 ΔIEC Mouse DSS group (Fut2) ΔIECDSS) and Fut2 ΔIEC mice R.man Gavage group (Fut2) ΔIEC DSS+Rh). After one week of adaptive feeding, in the DSS group and R.man Mice in the gavage group were given 2.5% (w / v) DSS dissolved in drinking water, which was changed every 2-3 days. Mice in the control group were given normal drinking water for 7 days. R.man The gavage group received 50 μL of 10 μL at regular intervals daily. 9 CFU R.man The bacterial suspension was resuspended in 150 μL of physiological saline and administered by gavage. The control group and the DSS group received the same amount, i.e., 200 μL of physiological saline, by gavage.

[0043] The mice's water intake, weight, and fecal characteristics were recorded at fixed times each day, and scores were assigned according to Table 1.

[0044] Table 1 Disease Activity Index Scoring Details

[0045] Mice were sacrificed on day 8 after DSS modeling. Mice were anesthetized with pentobarbital via intraperitoneal injection. The abdominal cavity was opened through a midline incision, and the colon was severed 1 cm above the anus. The intestinal segment was freed along the mesentery to the terminal ileum. The intestinal contents were emptied, and the entire colon was photographed and its length measured. Approximately 1 cm of the distal colon was soaked and fixed in 4% paraformaldehyde solution, followed by tissue embedding. The remaining portion was divided in half into proximal and distal colons, dried, and flash-frozen in liquid nitrogen, then stored at -80°C for subsequent RNA and protein extraction.

[0046] 3. Construction and evaluation of TNFα-sensitized mouse models 1) In the colitis induction and in vivo experiments, mice (6-8 weeks old) were treated with 2.5% DSS for 7 days. Simultaneously, the experimental group mice were administered oral gavage with DSS resuspended in 150 μL of physiological saline. R.man (10 9 CFU was dissolved in 50 μL. Other control groups were given an equal volume of physiological saline by gavage.

[0047] 2) In the assessment R.man In experiments sensitizing the efficacy of TNFα inhibitors, the TNFα inhibitor group received TNFα inhibitor treatment (5 mg / kg, intraperitoneal injection, Selleck, A2124) on day 3 after DSS treatment. Meanwhile, the TNFα inhibitor group received... R.man In addition to receiving the same TNFα inhibitor treatment, the group received oral gavage for 7 consecutive days. R.man .

[0048] 4. Colon tissue sections and hematoxylin-eosin staining 1) Intestinal tissue embedding and sectioning: The intestinal tissue was removed from the fixative, rinsed with water to remove the fixative, and then subjected to gradient dehydration in ethanol solution, followed by clearing with xylene. A small amount of semi-molten paraffin was first injected into a plastic embedding cassette, cooled, and then the tissue was placed inside for fixation. Paraffin was then added again to completely cover the tissue, and the cassette was cooled on a freezing stage. Serial sections were prepared using a microtome to a thickness of 6-10 μm. The sections were then mounted on clean glass slides, heated, spread, dehydrated, air-dried, and stored for subsequent experiments.

[0049] 2) Hematoxylin-eosin staining: a) Place the slices on a baking machine and bake at 65 ℃ for 1 h to dewax until hydrated. Then soak them in xylene I for 30 min, xylene II for 30 min, xylene III for 10 min, and then soak them in anhydrous ethanol I, 95% alcohol, 80% alcohol and 75% alcohol for 5 min each. Keep the slices moist throughout the process.

[0050] b) Then perform hematoxylin-eosin staining. Apply hematoxylin to the tissue and stain for 0.5-1 min, then rinse thoroughly with tap water, being careful not to let the water flow directly onto the tissue section; then add 1% hydrochloric acid alcohol to the tissue, and rinse with tap water after a few seconds; then add 1% ammonia solution to the tissue to achieve a blue return for about 1 min, rinse thoroughly with tap water, and then immerse the tissue in eosin staining solution for staining, and finally rinse once more with tap water.

[0051] c) Dehydration and mounting: Soak the sections in 75% alcohol, anhydrous ethanol, and xylene for 5 min each. Finally, mount with neutral resin.

[0052] 3) Microscopic observation and histopathological scoring: The slides were observed and photographed under an Olympus upright microscope IX71. Three slides from each mouse were selected for histopathological scoring, as detailed in Table 2 below: Table 2. Histopathological scores of acute colitis

[0053] 5. Immunofluorescence staining of colon tissue sections 1) After baking the sections in a 65 ℃ oven for 1 h, dewaxing is performed until hydration, following the same steps as hematoxylin-eosin staining.

[0054] 2) Antigen retrieval: Prepare citrate buffer (pH 6.0). Boil the buffer in an autoclave, then place the tissue sections in the buffer, ensuring they are completely submerged. Reheat the autoclave; once boiling and steaming, time for 2 minutes. Then rinse the autoclave under cold water for 25 minutes. After cooling, remove the sections and rinse them with PBS in citrate buffer, 5 minutes each time, for a total of 3 rinses. Shake off the liquid from the sections and run an immunohistochemical pen around the tissue. Disrupt the membrane with 0.3% Triton for 15 minutes, then rinse with PBS, 5 minutes each time, for a total of 3 rinses.

[0055] 3) Serum blocking: Prepare blocking solution with 10% goat serum or donkey serum, 3% BSA, and 0.3 mol / L glycine. Block at room temperature for 1 h to reduce non-specific staining.

[0056] 4) Primary antibody incubation: a) Prepare primary antibody: Add 20-30 μL of diluted antibody (ZO-1, Proteintech, Cat#21773-1-AP, RRID: AB_10733242) or (Occludin, Abclonal, Cat#A12621) to each tissue sample. Calculate the required amount of antibody according to the antibody dilution factor listed in the antibody instructions and prepare it with primary antibody dilution buffer.

[0057] b) Antibody incubation: Remove the blocking solution from the slides, do not rinse with PBS, and wipe off excess water with absorbent paper. Place the slides in a light-protected humidified chamber, drop the prepared primary antibody into the tissue circle, and incubate overnight at 4 ℃.

[0058] 5) Secondary antibody incubation: Warm to room temperature for 30 min. Rinse sections with PBS 3 times for 3 min each time. After shaking off excess water, carefully blot the sections with absorbent paper. Then, add the proportionally diluted fluorescent secondary antibody (488, Proteintech, RGAM002; 555, Proteintech, RGAM003; prepared according to the instructions and actual conditions) to the tissue. Incubate in a humidified chamber in the dark for 1 h, then rinse again with PBS.

[0059] 6) Nuclear staining: Add DAPI to the tissue, incubate in the dark for 10 min, and rinse with PBS for 3 min × 3 times.

[0060] 7) Mounting and observation: Blot off excess moisture and mount with anti-fluorescence quenching mounting medium, taking care to avoid air bubbles during mounting. Tissue sections can be stored at 4 ℃ and observed under a fluorescence microscope on the same day or the next day.

[0061] 6. Alcian blue staining of colon tissue sections 1) Sample acquisition and in situ fixation: In order to preserve the spatial structure of the intestinal mucus layer to the greatest extent, the colon tissue after ex vivo was not rinsed, and the distal segment of the mouse intestine was directly placed in Carnoy's fixative.

[0062] 2) Tissue processing and paraffin sectioning: After dehydration with graded ethanol and clearing with xylene, the tissue was embedded in paraffin and then cut into continuous sections with a thickness of 5 μm.

[0063] 3) Alcian blue staining: The sections were rehydrated with xylene and graded ethanol solutions. Then the sections were immersed in alcian blue solution at pH 2.5 for 15-30 minutes.

[0064] 4) Observe the intestinal mucus layer under a microscope.

[0065] 7. Bacterial fluorescence in situ hybridization / UEA-FITC co-staining 1) Tissue fixation and embedding: Freshly collected mouse distal colon samples were immediately immersed in in situ hybridization fixative and fixed overnight at 4 °C. The intestinal tissue was removed from the fixative, rinsed with water to remove the fixative, and then subjected to gradient dehydration in ethanol solution, followed by clearing with xylene. A small amount of semi-molten paraffin was injected into a plastic embedding cassette, cooled, and then the tissue was placed inside for fixation. Paraffin was then added again to completely cover the tissue, and the cassette was cooled on a freezing stage.

[0066] 2) Sectioning and Dewaxing: Continuous sectioning was performed using a microtome to a thickness of 6-10 μm. Sections were then mounted on clean glass slides, heated, spread, dehydrated, air-dried, and stored for subsequent experiments. Before starting the experiment, tissue sections were baked, then dewaxed three times with xylene for 5 minutes each time, followed by rehydration with a gradient of ethanol (100%, 85%, 75%), and finally equilibrated in sterile PBS.

[0067] 3) Bacterial probe hybridization: FISH detection was performed using a direct in situ hybridization kit for bacteria.

[0068] a) Pretreatment: The slides are first pretreated in the buffer provided in the kit to allow the bacterial cell wall to permeate.

[0069] b) Hybridization: The Roseburia-specific oligonucleotide probe (sequence SEQ ID NO. 1: 5'-TCAGACTTGCCG(C / T)ACCGC-3') labeled with the Cy3 fluorescent group at its 5' end was diluted in hybridization buffer. The probe-containing buffer was dropped onto tissue sections and incubated in a humidified chamber at 37 °C in the dark for 48 hours to ensure sufficient binding of the probe to the target RNA. After hybridization, the samples were washed with pre-warmed washing buffer under constant temperature conditions. The samples were then dried in a gradient of ethanol.

[0070] c) UEA-1 staining: Dilute FITC-labeled UEA I at a ratio of 1:200 and incubate with the slide at room temperature for 1 hour.

[0071] 5) Washing and mounting: Wash with preheated washing buffer at a constant temperature. Then, mount with a DAPI-containing anti-fluorescence quenching mounting medium to counterstain the host cell nucleus and bacterial DNA.

[0072] 6) Image Acquisition: Images were acquired using an Axio Observer 7 fluorescence microscope (Carl Zeiss, Germany) equipped with an Apotome module. Images were acquired in the Cy3 (red fluorescence, labeled Roseburia), DAPI (blue fluorescence, labeled nucleus), and FITC (green fluorescence, labeled fucose site) channels under the same exposure settings.

[0073] 8. Microbial DNA extraction and R.man Quantitative analysis 1) Total DNA extraction from colon tissue samples: Distal colon tissue samples (approximately 20-50 mg) were collected from mice, rapidly pre-cooled in liquid nitrogen, and then stored at -80°C. Total DNA from the tissue and intraluminal microorganisms was extracted using a DNA extraction kit (DP304, TIANGEN, Beijing, China). To ensure… R.man After the cell walls of Gram-positive bacteria were fully disrupted, the sample was transferred to a centrifuge tube pre-filled with 0.1 mm zirconia beads before routine lysis. The sample was mechanically disrupted using a tissue homogenizer at 60 Hz for 3 min. Subsequent steps strictly followed the kit instructions: digestion with proteinase K, specific binding to a silica gel column, and multiple washes, finally eluting the DNA with 50 μL of sterile elution buffer. DNA purity and concentration were measured using a NanoDrop 2000 spectrophotometer, ensuring an A260 / 280 ratio between 1.8 and 2.0, and the sample was stored at -20℃ for later use.

[0074] 2) R.man Routine PCR amplification and gel electrophoresis detection: a) For qualitative detection of colon tissue R.man Regarding the existence of the situation, R.man Primers were designed for the specific gene SBP (sequences are shown in Table 3 below). Amplification was performed using a reaction system constructed with a highly specific DNA polymerase.

[0075] Table 3 R.man Specific gene SBP primer details

[0076] b) PCR products were separated by 2% (w / v) agarose gel electrophoresis at a constant voltage of 110 V for 30 min. After electrophoresis, gel images were observed and acquired using a gel imaging system. The molecular weight of the amplified products was compared with that of the DNA marker to determine if it was the expected target band. The brightness of the target band was used for qualitative evaluation. R.man The colonization status of the gene in colon tissue and the degree of enrichment of the gene.

[0077] 3) A strict control system was also established in the experiment: with R.man DNA from the standard strain was used as a positive control; DNA from the colon tissue of mice that were not inoculated (control group) was used as a negative control; and an equal volume of sterile, enzyme-free water was used to replace the template DNA as a blank control (No-template control, NTC).

[0078] 9. Transmission electron microscopy 1) Sample fixation and preliminary treatment: The colon tissue was cut into small pieces and fixed in 2.5% glutaraldehyde for 2 hours.

[0079] 2) Post-fixation: The sample was then immersed in osmium tetroxide and kept at room temperature in the dark for 2 hours for post-fixation.

[0080] 3) Gradient dehydration and permeation: The sample was dehydrated using a series of gradient ethanol solutions and then immersed in pure resin overnight to ensure that the resin completely penetrated into the cells.

[0081] 4) Embedding and polymerization: The above sample was placed in a mold, filled with pure resin, and polymerized in a 60 ℃ constant temperature oven for 48 hours.

[0082] 5) Ultrathin sectioning: Performed using an ultrathin slicer.

[0083] 6) Transmission electron microscopy observation and imaging: Observation was performed using a Hitachi HT7800 transmission electron microscope.

[0084] 10. Extraction and concentration determination of intestinal tissue proteins 1) Tissue grinding: Using sterile scissors and forceps, cut tissue blocks the size of mung beans and place them in 1.5 ml centrifuge tubes. Add 3-5 1 mm zirconia grinding beads to each centrifuge tube, followed by 150-200 μL of RIPA lysis buffer (containing 1% protease inhibitor and 1% protein phosphatase inhibitor). Grind at 60 Hz for 120 s for 6-8 times until the tissue block is completely ground. If any visible tissue blocks remain, increase the number of grinding cycles.

[0085] 2) Protein Extraction: Place the tissue lysis buffer on ice and vortex 6 times for 15 seconds each time, with a 5-minute interval, for a total of about half an hour. Note that vortexing should ensure complete tissue lysis but not be prolonged, as this can generate heat and lead to protein degradation. During the vortexing intervals, keep the lysis buffer on ice. After vortexing, centrifuge at 12000 rpm for 15 minutes. The centrifuge should be pre-cooled at 4°C to prevent protein degradation. After centrifugation, pipette the supernatant into another clean centrifuge tube and record the volume. This is the tissue protein extract. Take 1 μL for protein concentration detection. Add 5× protein loading buffer (1 / 4 the volume of the protein extract) to the remaining samples and mix thoroughly. Denature at 95°C for 10 minutes in a PCR instrument, then aliquot and store at -80°C for long-term storage.

[0086] 3) BCA method for protein concentration determination a) Prepare the BCA working solution: Mix solution A (BCA reagent) and solution B (copper sulfate solution) from the kit thoroughly at a volume ratio of 50:1. Each sample requires 200 μL of working solution. Calculate the total required working solution volume: (8 standards + number of samples + 1 tube consumption) × 200 μL. Prepare as needed, just before use.

[0087] b) Preparing protein standard wells: Add 25 μL of PBS solution to 8 wells of a 96-well plate as standard wells, and add 24 μL of PBS solution to the remaining sample wells. Pipette 25 μL of the standard protein solution (4 mg / mL) into the first standard well and mix thoroughly to obtain a protein concentration of 2 mg / mL. Pipette 25 μL of the mixture from the first well into the second standard well, resulting in a protein concentration of 1 mg / mL. Then, sequentially dilute each standard well serially. The eighth well is left untreated. Following this procedure, eight standard wells with concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0 mg / mL were obtained.

[0088] c) Sample loading and detection: Add 1 μL of sample solution to each well of the sample to be tested. Then add 200 μL of BCA working solution to both the standard wells and the sample wells, and mix thoroughly by pipetting, taking care not to create air bubbles. Incubate the 96-well plate at 37 °C for approximately 15 min. After observing the color change of the standard wells according to the concentration gradient, measure the absorbance using a microplate reader. Set the wavelength of the microplate reader to 570 nm according to the instructions. Record the absorbance value of each well. If the absorbance value is low, the reaction time can be increased or the reaction can be carried out at 60 °C.

[0089] d) Calculation of protein concentration in the test sample: Plot a standard curve with the gradient protein content (mg / ml) of the standard wells on the x-axis and the measured absorbance values ​​on the y-axis. If the R-squared value is greater than 0.99, the standard curve is reliable and can be used for the next calculation. Calculate the corresponding protein concentration by comparing the absorbance values ​​of the test sample wells with the absorbance values ​​of the test sample wells, and then multiply it by the sample dilution factor to obtain the actual protein concentration of the sample.

[0090] 11. Western blot 1) Prepare SDS-PAGE gel a) Select the appropriate concentration of SDS-PAGE lower gel based on the molecular weight of the target protein in Table 4 below.

[0091] Table 4. Correspondence between SDS-PAGE separating gel concentration and optimal protein molecular weight separation range

[0092] b) Gel Preparation: Wash the gel casting glass plate and comb teeth with clean water. Clamp the glass plate onto the gel casting apparatus, applying force from both sides simultaneously. Ensure the gel casting apparatus scale matches the thickness of the glass plate interlayer. Add ddH2O between the glass plates, checking and confirming that the glass plates are aligned and clamped tightly to prevent gel leakage. Gently pour out the ddH2O from the apparatus and blot dry with filter paper. For example, to prepare a 1.5 mm thick gel in one step, mix 4 ml each of the lower gel solution and lower gel buffer. Then add 80-100 μL of coagulant, quickly pipette to mix again, and carefully pour the mixture along the sidewall into the gel casting glass plate until the lower gel level is approximately 1.5 cm from the top edge of the concave glass plate, leaving space for the upper gel. Gently shake the gel casting apparatus to level the lower gel solution. Take 2 ml each of the upper gel solution and upper gel buffer, mix them, add 20-40 μL of coagulant, and mix again. Slowly pour the mixture along the side wall of the gel casting plate, avoiding direct impact on the lower gel layer. Then, insert the comb teeth into the glass plate with the smooth side facing out, and use a pipette to remove any excess gel that overflows from the gel casting apparatus. Let it stand and wait for the gel to solidify. The solidification status can be judged by whether the liquid level is horizontal or whether any remaining excess gel has solidified. Finally, remove the comb teeth, and the gel is ready for electrophoresis.

[0093] 2) Protein loading and electrophoresis a) The electrophoresis solution is prepared as shown in Table 5 below.

[0094] Table 5 Electrophoresis Solution Formulation

[0095] b) Protein loading: The loading volume for cells is 20-40 ug / well. For tissues, the loading volume can be increased as needed. Take the protein sample out of the freezer, place it on ice to thaw, and shake it slightly after thawing. Then, the sample can be loaded according to the calculated volume, leaving 3-4 ul of marker on each well.

[0096] c) Electrophoresis: At the beginning, adjust the voltage to a constant 80 V. After about 30 minutes, when the sample bands have reached the lower gel, the voltage can be increased to a constant 120 V. Electrophoresis is carried out for about 1 hour. When the target bands are clearly separated from the marker positions, electrophoresis can be stopped and subsequent operations can be performed.

[0097] 3) Transfer membrane a) Prepare the transfer solution according to Table 6 below.

[0098] Table 6 Transfer Buffer Formulation

[0099] b) Transfer: After electrophoresis, discard the electrophoresis solution, rinse away excess foam with water, remove the glass plate, and prepare to cut the gel in the transfer solution. Keep the gel moist at all times to prevent it from drying out. Cut a PVDF membrane to the size corresponding to the gel. Use tweezers to cut the membrane; do not touch it with your hands. Mark the membrane with a carbon pen to distinguish the front and back sides. Activate the PVDF membrane by immersing it in anhydrous ethanol for 15 seconds to 1 minute. Place four layers of thin filter paper and one layer of transfer sponge on each side of the sandwich transfer clamp, moisten them in the transfer solution, and remove any air bubbles. Then, following the "black gel, white membrane" principle, place the protein gel and PVDF membrane sequentially on the filter paper. Handle carefully to avoid air bubbles between the protein gel and PVDF membrane, which can affect transfer efficiency. Carefully close the transfer clamp and place it into the transfer tank according to the "black to black, white to red" orientation. Connect the electrodes, ensuring the positive and negative electrodes are correctly connected. Set the electrophoresis apparatus to a constant current of 350 mA, turn on the power, and observe fine air bubbles forming in the transfer rack; this indicates that the circuit is functioning correctly. During transfer, the transfer rack can be placed in an ice-water mixture to cool it down, preventing excessive heat generation from interfering with transfer efficiency. The specific transfer time depends on the size of the target protein and can be roughly estimated based on molecular weight plus 20 minutes.

[0100] 4) Blocking: After the transfer is complete, open the transfer clamp and you can observe that the marker on the protein gel has been transferred to the PVDF membrane. Take out the membrane and place it in the rapid blocking solution for 15-30 min, and shake it slowly for 30-40 revolutions.

[0101] 5) Primary antibody incubation: Prepare the corresponding primary antibody (FUT2, abclonal, A5721; SLC3A2, Proteintech, Cat No. 15193-1-AP) according to the antibody titer using antibody dilution buffer. The primary antibody can be recycled about 3 times. Place the blocked protein band into the primary antibody for incubation, with the protein side down to ensure full contact with the antibody. Incubate overnight at 4 ℃ with gentle shaking.

[0102] 6) Secondary antibody incubation: Remove the incubation box from the refrigerator and allow it to warm to room temperature with gentle shaking for 30 min to 1 h. Then, place the band in TBST (TBS: Tween 20 = 1000:1) and shake rapidly (approximately 100-120 rpm) to wash away excess antibody. Wash three times, 10 min each time, replacing the TBST halfway through. Next, incubate the band with the corresponding rabbit / mouse antibody (the secondary antibody can be diluted with TBST or recycled 3-5 times; α-Tubulin, Proteintech, Cat No. 11224-1-AP) at room temperature with gentle shaking for approximately 1 h.

[0103] 7) Exposure: Remove the bands from the secondary antibody and wash three times with TBST for 10 minutes each time. Prepare for exposure. Mix the ECL exposure reagents at a 1:1 ratio of solution A to solution B. Remove the protein bands from the TBST and gently wipe off excess wash solution on filter paper, keeping the membrane moist. Then place the bands in the exposure solution, protein side down, to ensure full contact. Incubate for approximately 1 minute. Remove the bands and place them in the center of the exposure plate, protein side up, ensuring proper alignment and avoiding air bubbles or wrinkles between the bands and the plate. Select the "chemiluminescence" mode on the exposure apparatus and set the resolution, sensitivity, exposure time, and other exposure parameters. Start the exposure and save the data for analysis. If multiple bands need to be exposed on the same membrane, recover the bands and wash them quickly with TBST for 5 minutes, then place them in antibody elution buffer and shake (60-80 rpm) for 15 minutes, followed by another 5-minute wash with TBST. Then rinse and block, followed by primary and secondary antibody incubation, following the same steps as above.

[0104] 8) Semi-quantitative analysis: Select a clearly visible band with appropriate exposure and import it into ImageJ. Set the image type to "8-bit", the measurement mode to "pixel", and the measurement content to "grayscale value", "area", and "average grayscale value". The ratio of the grayscale value of the target protein to that of the internal reference protein is the relative expression level of the protein.

[0105] 12. RNA extraction from intestinal tissue 1) Tissue grinding: Use sterile scissors and forceps to cut tissue blocks the size of mung beans and place them in 1.5 ml sterile centrifuge tubes. Add 3-5 1 mm zirconia grinding beads to each centrifuge tube, followed by 500 μL of Trizol. Grind at 60 Hz for 120 seconds 3 times until the tissue block is completely ground. If any tissue blocks are still visible to the naked eye, the number of grinding cycles can be increased.

[0106] 2) RNA extraction: a) Add 2 / 5 volume of RNase-free double-distilled water (200 μL) to the tissue lysis buffer obtained above, mix by inverting, and let stand at room temperature for 5 min.

[0107] b) Pre-cool in an ultra-high-speed centrifuge and centrifuge at 12000 g for 15 min.

[0108] c) After centrifugation, the solution should separate into layers. Transfer the upper aqueous phase (containing RNA) to a new sterile centrifuge tube, being careful not to aspirate the lower impurities.

[0109] d) Add an equal volume of isopropanol to a new centrifuge tube, invert to mix, and let stand at room temperature for 10 min.

[0110] e) Centrifuge at 12000 g for 10 min. A white precipitate will be visible on the centrifuge tube wall after the centrifuge is finished. Aspirate the supernatant and retain the white precipitate.

[0111] f) Add 500 μL of 75% ethanol to wash the RNA precipitate. Note that the anhydrous ethanol must be prepared using RNase-free double-distilled water. During washing, invert the centrifuge tube to suspend the precipitate, and avoid breaking the RNA precipitate to maximize the yield.

[0112] Centrifuge at 8000 g for 3 min and discard the supernatant.

[0113] h) Add 500 μL of 75% ethanol and wash the precipitate again following the steps above, discarding the supernatant.

[0114] i) Invert the centrifuge tube onto clean tissue paper to air dry any remaining solution. Then add an appropriate amount of RNase-free double-distilled water and repeatedly pipette or vortex to mix and dissolve the precipitate. Aliquot the resulting RNA solution and store it at -80 °C for long-term storage.

[0115] 3) RNA concentration detection RNA concentration was detected using a NanoDrop spectrophotometer.

[0116] a) Clean the detection base three times with RNase-free double-distilled water.

[0117] b) Add 1 μL of RNase-free double-distilled water to the detection base using a pipette, lower the sample arm, and perform a blank control.

[0118] c) Use a pipette to take 1 μL of the target sample and drop it onto the detection base. Gently lower the sample arm and start the software to detect the absorbance. RNA purity can be judged by OD260 / OD280; a ratio of 1.8-2.2 is generally considered to indicate high purity. After detection, wipe the detection base with clean, lint-free paper before testing the next sample.

[0119] 13. Reverse transcription and real-time quantitative PCR (Real-time PCR) 1) Reverse transcription a) Prepare the reverse transcription reaction system, mix well, and centrifuge the liquid to the bottom of the tube. Component reaction amount 5 × HiScript III qRT SuperMix 4ul 1 ug of extracted RNA Add ddH2O until the volume reaches 20 ul. b) Reverse transcription reaction Temperature Time 37 ℃ for 15 min 85 ℃ 5 sec Maintain at 4℃ The resulting cDNA product can be used for subsequent qPCR reactions or stored at -20 ℃.

[0120] 2) Real-time quantitative PCR a) Configure the qPCR reaction system Component reaction amount 2 × ChamQ SYBR qPCR Master Mix 10 ul Primer 1 (10 uM) 0.4 ul Primer 2 (10 uM) 0.4 ul cDNA 2 ul Add ddH2O until the volume reaches 20 ul. SYBR and primers, and ddH2O and cDNA can be pre-mixed according to the actual number of samples and indicators, and then added to the PCR plate in equal proportions during sample loading to reduce loading errors. Primer sequences are shown in Table 7 below.

[0121] Table 7 PCR Primer Details

[0122] b) qPCR reaction Step Objective Number of cycles Temperature Time First step pre-denaturation 1 95 ℃ 30 sec The second step of the cycle reaction is carried out at 40-95℃ for 3-10 seconds. 60 ℃ 10-30s ec The third step involves using the default melting curve acquisition program. c) qPCR result analysis Using β-actin / GAPDH as an internal control, each sample was tested in duplicate (inter-duplicate error < 0.5 was considered valid data). The mean CT values ​​for both the internal control and the target indicator were calculated: ΔCT = mean CT value of the target indicator - mean CT value of the internal control; ΔΔCT = experimental group - control group; absolute RNA expression level = 2 - ΔCT; relative RNA expression level = 2 - ΔΔCT. A single sharp peak in the melting curve indicates the specificity of the target product and represents valid data.

[0123] 14. Flow cytometry experiment on bacterial adhesion cells 1) Sample preparation and cell harvesting: After completing R.man After co-incubation (CFDA-SE pre-labeled, green fluorescent) with colonic epithelial cells (37 °C, 2 h), the culture medium was removed. The cells were then slowly washed three times with pre-warmed calcium- and magnesium-free PBS to thoroughly remove unbound free bacteria. Cells were then gently scraped off using a rubber scraper.

[0124] 2) Single-cell suspension preparation: Transfer the collected cell suspension to a 1.5 mL EP tube and gently pipette to disperse cell clumps. Then, filter the suspension through a 40 μm nylon filter into a flow cytometry tube.

[0125] 3) Flow cytometry data acquisition: Detection was performed using a BD FACSymphony A1 high-parameter flow cytometer.

[0126] 4) Data analysis: Processed using FlowJo V10 software.

[0127] 15. Far Western Blotting Experiment 1) Protein component acquisition a) Biotin labeling of host cell membrane surface proteins: Adherent cells were separated using a non-enzymatic dissociation solution. After thorough washing with ice-cold PBS, the cells were resuspended and the concentration was adjusted to 2.5 x 10⁻⁶. 7cells / mL. EZ-Link™ Sulfo-NHS-LC-Biotin reagent (1.0 mg per mL of suspension) was centrifuged at room temperature and reacted at 4 °C for 2 hours. The reaction was then terminated with PBS containing 100 mM glycine, and free biotin was removed by repeated washing. To maintain protein conformation, 100 μM oxidized glutathione (GSSG) was added to the final wash buffer.

[0128] b) Enrichment and concentration of cell membrane proteins: After biotin-labeling, cells were added to a lysis buffer containing 1% Triton X-100, 10% glycerol, 100 μM oxidized glutathione, and a protease inhibitor. After lysis with shaking on ice for 60 minutes, the supernatant was collected by high-speed centrifugation at 4 °C and 20,000 g. The supernatant was then replaced with 10 mM Tris (pH 7.5) using an ultrafiltration tube with a 3 kDa cutoff. Approximately 50 μL of concentrated protein sample was prepared. The non-biotin-labeled group served as an experimental control.

[0129] c) Extraction of bacterial surface proteins: Collect proteins from the logarithmic growth phase... R.man The bacterial strain was centrifuged at 8000 rpm for 10 min, and protein samples were prepared at a ratio of 1 g of bacterial cells to 3 mL of lysis buffer. The lysis buffer was prepared by dissolving 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl, and 1 mM PMSF in 300 μg / mL lysozyme solution. The bacterial culture was incubated at 4 °C with shaking for 30 min, followed by the addition of 1% Triton X-100 and sonication for 12 cycles, with each sonication lasting 15 s. Then, 10 mg / kg DNase I and 10 mM MgCl2 were added, and the culture was incubated at room temperature for 15 min to degrade nucleic acids. The culture was then centrifuged at 4 °C and 10000 g for 15 min, and the supernatant was collected and stored at -20 °C for later use.

[0130] 2) Electrophoretic separation and membrane transfer: Bacterial surface proteins were collected and separated using a 7.5% SDS-PAGE gel. The electrophoresis parameters were set to 80 V for an initial 20 minutes, and then increased to 125 V until the electrophoresis was completed after the proteins entered the separating gel. Subsequently, the proteins were transferred to a PVDF membrane using a wet transfer method (400 mA constant current, 2 h), and kept in an ice bath throughout the process to prevent heat denaturation.

[0131] 3) In situ refolding of membrane-attached proteins: To restore the protein conformation lost due to SDS treatment, a guanidine hydrochloride gradient dilution strategy was adopted to perform stepwise denaturation and refolding treatment on PVDF membrane-attached proteins in an AC buffer system, aiming to reconstruct the functional 3D domains that can bind to bait proteins.

[0132] 4) Blocking and binding: The membrane surface was blocked for 1 hour using PBST containing 5% skim milk powder. Subsequently, the membrane was immersed in binding buffer containing biotinylated cell bait protein (Bait) (concentration 0.05 μg / μL) and incubated overnight at 4°C.

[0133] 5) Affinity development and band recovery: After washing with PBST, incubate with HRP-streptavidin diluted 1:1,000 for 1 hour. Develop using an ECL-enhanced chemiluminescence kit. Based on the development location, accurately extract specific interaction bands from the corresponding SDS-PAGE gel for subsequent biomolecular mass spectrometry identification and analysis.

[0134] 16. Biotin pull-down test and protein identification 1) Protein components were obtained as described above.

[0135] 2) Magnetic bead pretreatment and bait protein capture: Take 50 μL of streptavidin magnetic bead suspension, place it on a magnetic rack, discard the preservation solution, and wash three times with ice-cold PBST (PBS containing 0.05% Tween-20). Resuspend the extracted biotinylated bait protein in 500 μL binding buffer and incubate with the magnetic beads at 4 ℃ for 4 hours by horizontal rotation. The high affinity between biotin and streptavidin securely anchors the bait protein to the magnetic bead surface.

[0136] 3) Co-incubation with target protein: After incubation, the magnetic beads were separated using a magnetic rack, and unbound bait proteins were removed by washing with PBST. The target protein was then added. The mixture was incubated overnight at 4 °C with slow inversion to allow the bait protein to fully bind to the target receptor protein.

[0137] 4) Elution and Sample Recovery: The following day, magnetic beads were collected using a magnetic rack and thoroughly washed with PBST to remove physically adsorbed proteins. 50 μL of 1× SDS-PAGE loading buffer was added to the magnetic beads, and the mixture was heated in a 95 °C metal bath for 5 minutes. The high-temperature denaturation caused the binding between streptavidin and biotin to break, simultaneously eluting the interaction complex from the magnetic beads.

[0138] 5) Electrophoretic separation and mass spectrometry compatible silver staining: Collect the elution supernatant and perform SDS-PAGE electrophoresis. After electrophoresis, staining is performed using a mass spectrometry compatible silver staining kit.

[0139] 6) Band recovery and identification: In a clean bench, the experimental group and the blank control group (with magnetic beads but no bait protein) were compared. The specific protein bands that appeared in the experimental group were accurately extracted for subsequent biomolecular mass spectrometry identification and analysis.

[0140] 17. Identification of protein gel strips using liquid chromatography-mass spectrometry (LC-MS) 1) Visualization of protein bands a) Coomassie Brilliant Blue Staining Method: After SDS-PAGE electrophoresis separation, take the gel block containing the target protein and rinse thoroughly with ultrapure water. Then, completely immerse the gel in Coomassie Brilliant Blue staining solution and destain at room temperature on a horizontal shaker. Once the target protein band is clearly visible, remove the staining solution and rinse repeatedly with ultrapure water until the background color completely disappears, ensuring a clear contrast between the protein band and the gel matrix for accurate gel cutting.

[0141] b) Mass spectrometry compatible silver staining method: After SDS-PAGE electrophoresis, remove the gel and place it in a clean container. First, fix it with fixative for 30 min. Then wash twice with 30% ethanol to remove acid. Add sensitizing solution and soak for 30 min, then rinse thoroughly with ultrapure water. Place the gel in silver nitrate solution for staining in the dark for 20 min. After staining, add developing solution until the target protein band is clearly visible, and immediately add stop solution to stop the reaction.

[0142] 2) In-situ trypsin digestion and peptide extraction: Target protein bands were excised and destained. Then, proteomics-grade sequence-modified trypsin was added for in-situ digestion. The digestion products were recovered using a specific peptide extraction buffer and incubated at 37 °C for 30 minutes with 15 minutes of sonication to maximize peptide extraction from the gel. The supernatant was collected by centrifugation, and the extracts were combined and dried using a vacuum centrifuge.

[0143] 3) Nano-liquid chromatography-mass spectrometry (NLC-MS / MS): The dried peptide sample was dissolved in Nano-LC mobile phase A. The sample was loaded onto a nanoViper C18 trapping pre-column (3 μm, 100 Å) via an autosampler for online desalting and enrichment, and then entered the analytical column for high-resolution separation. The sample was then coupled online with a mass spectrometer for secondary mass spectrometry acquisition.

[0144] 4) Bioinformatics Retrieval and Protein Identification: The raw mass spectrometry data (.wiff format) was converted and peak lists were extracted using ProteoWizard (v3.0.10577) software to generate a standard .mgf format file. This file was then submitted to the Mascot (v2.3.02) search engine for matching searches in the relevant species database. The reliability of protein identification was evaluated using the Protein Score CI %; a confidence level exceeding 95% was considered statistically significant.

[0145] 18. GST pull-down assay and protein identification 1) Expression and purification of recombinant proteins: Proteins derived from *Reuteria reuteri* (…) Human rosebuds The SBP gene (site tag: RHOM_06615) of strain DSM16839T was inserted and ligated into the pGEX-6P-1 vector. The resulting recombinant plasmid was then transformed into *E. coli* BL21 (DE3) competent cells. When the bacterial culture reached an optical density (OD600) of 0.8, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and protein synthesis was induced at 25 °C for 16 hours. The bacteria were then collected by centrifugation and resuspended in lysis buffer containing 1 mg / mL lysozyme. After cell lysis and centrifugation, the supernatant was collected and co-incubated with GST-tagged magnetic agarose gel beads to enrich the GST-SBP fusion protein. Finally, the purified GST-SBP protein was obtained by elution.

[0146] The SBP protein sequence can be found at: https: / / www.uniprot.org / uniprotkb / G2T2Q7 / entry The SBP amino acid sequence can be found at: https: / / www.ncbi.nlm.nih.gov / nuccore / CP003040.1?from=1456827&to=1458260 pGEX-6P-1 vector sequence: https: / / www.ncbi.nlm.nih.gov / nuccore / U78872.1 2) Screening of interacting membrane proteins: To screen membrane proteins that interact with SBP, membrane proteins extracted from HT29 cells were incubated overnight at 4 °C with recombinant GST-SBP protein. GST magnetic beads were then added and incubation continued for 4 hours to capture the protein complex. After thorough washing, the magnetic beads were placed in 40 μL of protein loading buffer and boiled. The samples were then subjected to SDS-PAGE electrophoresis and silver staining. Interacting proteins were identified by mass spectrometry.

[0147] 3) Validation of SBP-SLC3A2 interaction: To validate the interaction between SBP and SLC3A2, total protein from HT29 and SW480 cells was incubated with GST-SBP protein overnight at 4 °C. A portion of the mixture was collected as an input sample to characterize the basal expression levels of GST-SBP and SLC3A2. The remaining sample was incubated with GST-tagged magnetic agarose gel beads for 4 hours. Western blot analysis of the eluted protein complexes was performed using an anti-SLC3A2 specific antibody to detect co-precipitated proteins.

[0148] II. Experimental Results 1. R.man Planting exerts anti-inflammatory and barrier protective effects, and the extent of these effects is related to the level of planting. ① An acute colitis model in mice was established using sodium dextran sulfate (DSS), and the experimental animals were administered... R.man Gavage treatment ( Figure 1 A). Phenotypic observation showed that intervention with this bacterium effectively improved weight loss in some of the tested mice. Figure 1 B), while lowering the Disease Activity Index (DAI) level ( Figure 1 C). Pathological examination results showed that the infiltration of inflammatory cells in the intestines of the intervention group was significantly reduced, and the severity of lesions such as crypt destruction and abscess formation was significantly alleviated. The pathological score and the condition of colonic shortening were both significantly improved. Figure 1 DG). But R.man Gavage had limited effect on improving the condition of individuals with significant weight loss. These results combined suggest that... R.man The anti-inflammatory effect exhibited significant individual variability, showing a significant effect only in a subset of mice, while having no noticeable effect in another subset. Based on the Disease Activity Index (DAI) and intestinal pathology score, [the mice were further analyzed]. R.man The gavage group (WT DSS+Rh) was divided into an effective subgroup and an ineffective subgroup. Wild-type control (WTCon), model group (WT DSS), and two subgroups of mice were selected, and inflammatory factors and intestinal barrier-related molecules were detected. qPCR results showed that compared with the WT DSS group, the effective subgroup had significantly lower levels of pro-inflammatory factors and significantly higher expression of intestinal barrier-related molecules. Figure 1 H).

[0149] ② R.man Its anti-inflammatory and barrier-protective effects may be related to its colonization level in the gut. On day 8 of the experiment, the intestinal contents of mice were examined. R.manThe colonization level. Fluorescence in situ hybridization detection revealed that the colonization abundance of this bacterium in the intestines of mice in the effective intervention group (WT DSS+Rh Effective) (significantly relieved inflammation) was significantly higher than that in the ineffective group (WT DSS+Rh Ineffective) (no significant improvement in inflammation). Figure 2 A) Intestines R.man PCR detection of DNA showed that the effective group exhibited specific amplification bands, indicating successful colonization and stable detection of the bacteria; the ineffective group showed weak or absent amplification bands, making effective detection of the target strain impossible. Figure 2 B). The qPCR quantitative analysis results are consistent with the above detection conclusions. Figure 2 C). Summary of the above suggestions R.man The anti-inflammatory and barrier-protective effects may be related to the level of intestinal colonization, which may explain the heterogeneity of clinical probiotic efficacy.

[0150] 2. Effects of Fut2 on intestinal epithelial fucoidylation R.man Planting Stable Fut2 knockdown and overexpression cell lines were constructed in vitro, and validation was performed at the protein and transcriptional levels using Western blot and qPCR techniques, respectively. Figure 3 EF). Subsequent in vitro bacterial adhesion experiments showed that downregulation of Fut2 expression significantly inhibited bacterial adhesion; competitive blocking of the α1,2-fucose site in intestinal epithelium by UEA-I also significantly reduced bacterial adhesion. Conversely, overexpression of Fut2 enhanced bacterial adhesion, and this pro-adhesion effect could be reversed by UEA-I blockade. Figure 3 AB). Quantitative analysis by flow cytometry further corroborated the above conclusions. Figure 3 CD). The above results indicate that Fut2 can mediate α1,2-fucosylation modification in intestinal epithelial cells. R.man The colonization process was described. The impact of host genetic inheritance on the gut microbiota was elucidated, providing new targets for personalized probiotic therapy for patients with different genotypes.

[0151] Table 8 Fut2 knockdown sequence

[0152] Table 9 Fut2 overexpression sequences

[0153] 3. R.man Surface sugar ABC transporter substrate-binding protein SBP may be involved in bacterial colonization. Using biotinylated HT29 membrane protein as a probe, screening was performed using Far-western blotting technology. R.manMembrane proteins ( Figure 4 A). After cutting the target band from the gel, the protein components were identified by liquid chromatography-mass spectrometry, and a total of 4 candidate proteins were obtained. Figure 4 B). Biotin Pull-down technology was used for screening concurrently. R.man Membrane proteins ( Figure 4 C), and mass spectrometry identification also yielded 4 candidate proteins (C). Figure 4 D). Intersection analysis of the two sets of mass spectrometry data revealed that the sugar ABC transporter substrate-binding protein (SBP) was successfully identified in both independent experiments.

[0154] 4. R.man Surface SBPs can bind to SLC3A2 on the intestinal epithelial surface, mediating bacterial colonization. ① SLC3A2 in intestinal epithelium may play an important role in bacterial adhesion. High-purity GST-SBP recombinant protein was obtained by purifying with E. coli and GST-tagged magnetic beads. Figure 5 A). Subsequently, the extracted HT29 cell membrane protein fraction was co-incubated with GST-SBP protein, and the potential protein complex was captured using GST pull-down technology. After separation by SDS-PAGE electrophoresis and silver staining, differential bands were extracted for mass spectrometry identification. The results suggested that SLC3A2, a member of the solute carrier family, may be a key candidate target protein for SBP interaction. Figure 5 BC).

[0155] ② R.man Surface SBPs can bind to SLC3A2 on the intestinal epithelial surface, mediating bacterial colonization. In HT29 and SW480 cells, stable SLC3A2 knockdown and overexpression cell lines were constructed, and Western blot and qPCR were used to validate the expression at the protein and transcriptional levels, respectively. Figure 6 AB). Subsequent in vitro bacterial adhesion experiments showed that SLC3A2 knockout significantly reduced bacterial adhesion ability, while overexpression of SLC3A2 significantly enhanced bacterial adhesion levels. Figure 6 C). In summary, R.man The surface SBP protein can bind to the intestinal epithelial cell surface receptor SLC3A2, thereby mediating the effective colonization of bacteria in the gut.

[0156] The SLC3A2 knockdown vector sequence can be found at: https: / / www.vectorbuilder.cn / vector / VB241012-1136mqj.html The SLC3A2 overexpression vector sequence can be found at: https: / / www.vectorbuilder.cn / vector / VB241012-1137vty.html 5. Fut2 regulates intestinal epithelial fucoidylation modification. R.man Colonization plays an anti-inflammatory and barrier protective role In in vitro experiments, several mouse group models were constructed, including: wild-type normal control group (WT Con), intestinal epithelial cell Fut2-specific knockout control group (Fut2...). ΔIEC Con), wild-type colitis induction group (WT DSS), Fut2 knockout colitis induction group (Fut2) ΔIEC DSS), and based on this, give R.man The intervention treatment group (WT DSS+Rh and Fut2) ΔIEC DSS+Rh)( Figure 7 ) Administered in wild-type mice R.man After intervention, the weight loss trend caused by DSS modeling was effectively suppressed. Figure 8 A), while significantly reducing the disease activity index ( ). Figure 8 B), and it has a significant effect on improving the condition of shortened colon length. Figure 8 CD). Pathological histological examination results showed that the degree of inflammatory infiltration in the intestinal tissue of mice treated with WT DSS+Rh was significantly reduced, the occurrence of crypt morphology abnormalities and abscesses was significantly reduced, and mucosal tissue damage was also significantly repaired. Figure 8 EF). Immunofluorescence assay results showed that, R.man It can effectively reverse the suppressed expression of tight junction-related proteins ZO-1 and Occludin in WT DSS model mice, thereby strengthening the integrity and defense capabilities of the intestinal barrier. Figure 9 ).

[0157] 6. R.man Surface SBP can exert anti-inflammatory and barrier protective effects. An intestinal epithelial cell inflammation model was induced using TNFα, and exogenous SBP protein was administered. qPCR results showed that SBP intervention could downregulate inflammatory cytokine expression and exert a protective barrier function. Figure 10 ).hint R.man The surface SBP can exert anti-inflammatory and barrier protective effects.

[0158] 7. R.man Mediating anti-inflammatory and intestinal barrier protective effects can sensitize the efficacy of TNF-α inhibitors. With Fut2 ΔIEC A multi-factor combined intervention animal model was established using mice and wild-type mice. Experimental animals were randomly divided into 8 groups according to treatment: wild-type blank control group, Fut2... ΔIEC Blank control group, wild-type DSS model group, Fut2 ΔIEC DSS model group, wild-type DSS combined with TNFα inhibitor treatment group, Fut2 ΔIEC The DSS combined with TNFα inhibitor treatment group, and additional additive treatment R.man The wild-type DSS + TNFα inhibitor + Rh combination treatment group underwent intervention. (Compared to Fut2) Δ IEC DSS + TNFα inhibitor + Rh group ( Figure 11 The results confirmed that... R.man Combined treatment significantly enhanced the therapeutic effect of TNF-α inhibitors on colitis, and the body weight of mice recovered. Figure 12 A) DAI score decreased ( Figure 12 B) The shortening of colon length was improved, and the histopathological damage score was also significantly reduced. Figure 12 (CE). However, in Fut2 knockout mice, the above-mentioned inflammation-related phenotypes did not show significant remission. This further explains... R.man Its surface SBP protein can sensitize the anti-inflammatory and barrier protective effects of TNF-α inhibitors. Combined with the results of fluorescence in situ hybridization and real-time quantitative PCR detection in colon tissue, it can be seen that, compared with Fut2 knockout mice, the intestinal tract of WT mice treated with bacterial culture via gavage... R.man Both colonization abundance and total bacterial count showed a significant increase. Figure 13 In summary, this explains... R.man The SBP protein on its surface can sensitize the anti-inflammatory and barrier protective effects of TNF-α inhibitors, suggesting that this probiotic or SBP protein may serve as an adjunct therapy for TNF-α inhibitors, enhancing efficacy and reducing adverse reactions while reducing drug dosage.

[0159] In summary, this invention confirms R.man The surface SBP protein can exert anti-inflammatory and barrier protective effects and sensitize TNFα inhibitors to alleviate colitis under Fut2 fucosylation modification.

[0160] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Roseburia hominis Its application in the preparation of products for the prevention and treatment of inflammatory bowel disease is characterized by, The product includes Roseburia hominis The product is a live bacterium or its surface glyco-ABC transporter substrate-binding protein SBP, and is intended for use in subjects with inflammatory bowel disease in whom the level of intestinal epithelial fucosylation is not significantly reduced.

2. The application according to claim 1, characterized in that, Those whose intestinal epithelial fucosylation levels were not significantly reduced included subjects whose FUT2 gene was functionally wild-type.

3. The application according to claim 1, characterized in that, The product also contains a TNF-α inhibitor.

4. The application according to claim 1, characterized in that, The product is used in combination with TNF-α inhibitors.

5. The application according to any one of claims 3-4, characterized in that, The product is used to enhance the anti-inflammatory and / or intestinal barrier protective effects of TNF-α inhibitors.

6. The application according to claim 5, characterized in that, The TNF-α inhibitors include anti-TNF-α antibodies.

7. The application according to claim 1, characterized in that, The SBP protein mediates by binding to SLC3A2 on the surface of intestinal epithelial cells. Roseburia hominis Intestinal colonization.

8. The application according to claim 1, characterized in that, The inflammatory bowel disease includes ulcerative colitis.

9. The application according to claim 1, characterized in that, The Roseburia hominis include Roseburia hominis strain A2-183T.

10. The application according to claim 1, characterized in that, The products include pharmaceuticals.

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