Use of bacteroides fragilis for preventing and treating celiac disease

Abstract

The application discloses application of Bacteroides fragilis in preparation of products for preventing and / or treating celiac disease, and a composition containing Bacteroides fragilis for treating and / or preventing celiac disease. The application proves through a large number of experiments that Bacteroides fragilis, especially Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685, can increase the expression of tight junction related proteins, improve intestinal barrier permeability, down-regulate TTG-IgA antibodies and pro-inflammatory factors IFN gamma and IL-15, reduce inflammation, and effectively prevent and treat celiac disease.

Description

Technical Field

[0001] This invention relates to the application technology of Bacteroides fragilis in the prevention and treatment of celiac disease, and in particular to the application of a strain of Bacteroides fragilis ZY-312 in the prevention and treatment of celiac disease. Background Technology

[0002] Celiac disease (CD), also known as gluten-sensitive enterocolopathy, is a chronic, multi-organ autoimmune disease affecting the small intestine in children and adults, particularly in genetically susceptible individuals expressing the HLA-DQ2 / -DQ8 gene. It is triggered by the intake of gluten-containing foods, leading to atrophy of the small intestinal mucosa and manifesting primarily as chylous diarrhea. Clinical manifestations include chronic diarrhea, abdominal discomfort, weight loss, and anemia. Extraintestinal manifestations may also occur, such as herpetic dermatitis and peripheral neurological symptoms. Previously considered a rare disease, increased understanding of celiac disease in recent years, the introduction of highly sensitive serological screening techniques, the application of small intestinal biopsy, and the development of gene testing methods have revealed that celiac disease affects approximately 1% of the global population, and this trend is gradually increasing.

[0003] Lifelong gluten-free diet (GFD) is currently the only universally recognized effective treatment. Most patients experience gradual symptom relief or disappearance after adhering to a gluten-free diet for several days to weeks, and serum markers gradually return to normal after several weeks to months. Intestinal mucosal recovery is relatively slow, and complete histological restoration is very rare. Although the benefits of GFD outweigh the risks for patients, it is difficult to avoid in daily life. Even with consistent GFD adherence, approximately 60% of celiac disease patients do not show significant improvement in small intestinal mucosal pathology, their gut microbiota cannot be fully restored, and intestinal symptoms persist. Furthermore, a gluten-free diet can lead to nutritional imbalances, resulting in insufficient intake of certain micronutrients such as iron, folic acid, vitamin B1, and vitamin B12.

[0004] Currently, there is no effective drug treatment for celiac disease. However, as the immunopathogenesis of celiac disease is gradually understood, some new treatment methods are undergoing clinical trials. Microbial therapy is one such novel treatment for celiac disease and has been extensively studied in recent years. Glycol, the main component of gluten, cannot be completely degraded by digestive enzymes in the gastrointestinal tract, generating peptides rich in glutamine and proline. This triggers excessive adaptive and innate immune responses in the gut, damaging the intestinal mucosal structure and leading to the disease. Numerous studies have demonstrated that the gut microbiota can modulate immunity and influence intestinal permeability by regulating the tight junctions of the intestinal epithelium. For example, Bifidobacteria can reduce the increase in intestinal epithelial permeability induced by gluten stimulation, downregulate the Th1 pathway in celiac disease, thereby reducing inflammation and histological lesions in the jejunum.

[0005] Bacteroides fragilis (B. fragilis) is a Gram-negative, rod-shaped, blunt-ended, darkly stained, capsule-bound, non-spore-forming, non-motile obligate anaerobic bacterium. It is divided into enterotoxigenic (ETBF) and non-enterotoxigenic (NTBF) types. It is part of the normal intestinal flora of humans and animals and normally resides in the mucous membranes of the human respiratory tract, gastrointestinal tract, and urogenital tract. The applicant's research team began exploring and isolating Bacteroides fragilis (strain ZY-312) from the feces of healthy infants in 2012. Studies have shown that ZY-312 has protective effects on the intestinal mucosa and intestinal barrier and regulates immunity (Fan H, Chen Z, Lin R, et al. Bacteroides fragilis Strain ZY-312 Defense against Cronobacter sakazakii-Induced Necrotizing Enterocolitis In Vitro and in a Neonatal Rat Model. mSystems. 2019;4(4):e00305-19. Published 2019 Aug6.), and has great potential in the prevention and treatment of intestinal diseases.

[0006] The Bacteroides fragilis ZY-312 strain used in this invention does not contain the BFT gene and is a non-toxin-producing strain. Acute toxicity tests have confirmed that this strain is non-pathogenic to normal mice and nude mice (Wang Y, Deng H, Li Z, Tan Y, Han Y, Wang X, Du Z, Liu Y, Yang R, Bai Y, Bi Y, Zhi F. Safety Evaluation of a Novel Strain of Bacteroides fragilis. Front Microbiol. 2017 Mar 17;8:435.). According to patent ZL201510459408.X and scientific literature Xu W, Su P, Zheng L, Fan H, Wang Y, Liu Y, Lin Y, Zhi F. In vivo Imaging of a Novel Strain of Bacteroides fragilis via Metabolic Labeling. Front Microbiol. 2018 Oct 1;9:2298., this strain has good tolerance to gastric acid and bile salts, ensuring its survival and effective colonization in the stomach.

[0007] Although studies have shown that gut microbiota has great potential in preventing and treating celiac disease, there are currently no microecological preparations specifically developed for the prevention and treatment of celiac disease. Therefore, it is necessary to explore the application of Bacteroides fragilis in the prevention and treatment of celiac disease. Summary of the Invention

[0008] To overcome the aforementioned deficiencies in the prior art, the purpose of this invention is to provide an application of *Bacteroides fragilis* in the preparation of products for the prevention and / or treatment of celiac disease. Extensive experimental evidence demonstrates that *Bacteroides fragilis*, particularly the strain with accession number CGMCC No. 10685, can effectively prevent and treat celiac disease by protecting the intestinal mucosa and intestinal barrier, regulating immunity, and reducing inflammation.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of Bacteroides fragilis in the preparation of products for the prevention and / or treatment of celiac disease, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312 with accession number CGMCC No. 10685.

[0010] In some implementations, the Bacteroides fragilis is a live or inactivated bacterium.

[0011] In some implementations, the inactivated bacteria are either morphologically intact or morphologically incomplete.

[0012] In some embodiments, the *Bacteroides fragilis* is one or more of the following: live *Bacteroides fragilis* cells, *Bacteroides fragilis* cells that have been inactivated, genetically recombined, modified, altered, attenuated, chemically treated, or physically treated; *Bacteroides fragilis* lysate; and *Bacteroides fragilis* liquid culture supernatant.

[0013] In some implementations, celiac disease includes typical celiac disease, atypical celiac disease, or asymptomatic celiac disease.

[0014] In some implementations, the product is a drug.

[0015] In some embodiments, the drug also includes a pharmaceutically acceptable carrier and / or excipients.

[0016] In some embodiments, the dosage form of the drug is pills, tablets, powders, granules, capsules, solutions, tube feeding preparations, suspensions, creams, sprays, oral liquids, enemas, ointments, or patches.

[0017] In some implementations, the drug is administered intermittently, periodically, continuously, or over a long period of time.

[0018] In some implementations, the drug is administered orally or via enema.

[0019] In a second aspect, the present invention provides the use of a composition containing Bacteroides fragilis in the preparation of products for the treatment and / or prevention of celiac disease, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312 with accession number CGMCC No. 10685.

[0020] In some embodiments, the composition is a pharmaceutical composition.

[0021] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipients.

[0022] The beneficial effects of this invention are: This invention demonstrates through numerous experiments that Bacteroides fragilis, particularly Bacteroides fragilis ZY-312 with accession number CGMCC No. 10685, can effectively prevent and treat celiac disease by protecting the intestinal mucosa and intestinal barrier, regulating immunity, and reducing inflammation. Attached Figure Description

[0023] Figure 1 This is a colony characteristic diagram of Bacteroides fragilis ZY-312 from Example 1 of the present invention; Figure 2 Microscopic observation of Bacteroides fragilis ZY-312 after Gram staining in Example 1 of this invention. Detailed Implementation

[0024] The microbial strain used in the implementation of this invention was deposited on April 2, 2015, at the China General Microbiological Culture Collection Center (CGMCC) (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing). Classification and naming: *Bacteroides fragilis* ZY-312, accession number CGMCC No. 10685. *Bacteroides fragilis* ZY-312 was isolated and obtained by the applicant and is already protected by an authorized patent (patent number 201510459408.X).

[0025] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0026] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available. All cells were purchased from ATCC; all cell culture materials were purchased from Gibco; and all laboratory animals were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.; or they can be prepared by known methods. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0027] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] Example 1: Fermentation culture of Bacteroides fragilis Bacteroides fragilis ZY-312 was streaked onto blood agar plates and anaerobic incubated for 48 hours. Colony morphology, staining characteristics, size, coccoid shape, and distribution were observed.

[0029] Colony characteristics: After 48 hours of incubation on blood agar plates, Bacteroides fragilis ZY-312 colonies are round, slightly convex, translucent, white, with a smooth surface, and do not hemolyze. The colony diameter is between 1-3 mm. See [link to relevant documentation]. Figure 1 .

[0030] Microscopic morphology: Gram-stained Bacteroides fragilis ZY-312 is a Gram-negative bacterium, exhibiting a typical rod shape with blunt, rounded, and deeply stained ends. The unstained portion in the middle of the cell resembles a vacuole. (See also...) Figure 2 .

[0031] Select a single colony and inoculate it into plant-derived peptone liquid medium for fermentation culture for 8 hours (temperature 37℃). Centrifuge the obtained bacterial solution at 3000 r / min for 15 min, remove the supernatant, and collect the precipitate to obtain Bacteroides fragilis ZY-312 bacterial sludge.

[0032] Example 2 Preparation of live Bacteroides fragilis culture 1) Streak Bacteroides fragilis ZY-312 strains were inoculated onto blood agar plates and cultured anaerobically at 37°C for 48 hours.

[0033] Colony characteristics: After culturing Bacteroides fragilis ZY-312 on blood agar plates for 48 hours, the colonies are round, slightly convex, translucent, white, smooth, and non-hemolytic, with a diameter of 1-3 mm.

[0034] 2) Enrichment: Select a single colony from step 1) and inoculate it into TSB (tryptone soybean broth containing 5% fetal bovine serum) for enrichment culture. Store the resulting bacterial solution for later use.

[0035] 3) Bacteroides fragilis ZY-312 live bacterial suspension: The bacterial suspension prepared in step 1) was analyzed for bacterial count using McFarland turbidimetric tubes, and diluted to 10⁻⁶ with physiological saline. 8 CFU / mL and 10 10 CFU / mL, store for later use.

[0036] 4) Prepare 10 μL of live Bacteroides fragilis ATCC25285 culture using the method described above. 10 CFU / mL, stored for later use. Example 3: Preparation of inactivated Bacteroides fragilis powder (1) Centrifuge the fermentation broth of Bacteroides fragilis prepared in Example 1, collect the wet cells, add physiological saline at a ratio of cells:physiological saline = 1:(10~30)(m:v) to resuspend and wash the bacterial sludge, and centrifuge again to collect the washed cells.

[0037] (2) Add 5% maltodextrin + 0.9% sodium chloride mixture to the bacterial cells obtained in step (1) according to the ratio of bacterial cells: excipient = 1: (5~15) (m:m). After stirring and dispersing, heat inactivate at (70~100)±5℃ for (20~40)±5 minutes to obtain inactivated bacterial solution.

[0038] (3) Centrifuge the inactivated bacterial solution obtained in step (2) to collect the inactivated bacterial sludge.

[0039] (4) Add excipients to the inactivated sludge collected in step (3) so that the total weight is consistent with the weight of the bacterial solution before inactivation, stir until completely dissolved, and obtain the inactivated bacterial stock solution.

[0040] (5) The inactivated bacterial stock solution obtained in step (4) is subjected to vacuum freeze drying. After pre-freezing at -40±2℃ for 1~3 hours, it is pre-freezing at -20±2℃ for 0.5~1 hours, and finally pre-freezing at -40±2℃ for 0.5~2 hours. After drying once (at -5±2℃ and 0±2℃) and desorption drying (at 35±2℃) under a vacuum of 0.25mbar, inactivated bacterial powder is prepared. The bacterial count of the bacterial powder reaches 1×10 11 Cell / g.

[0041] In the experiment, 1×10⁶ Bacteroides fragilis ZY-312 bacterial suspensions were prepared using bacterial powder. 8 Cell / mL and 1×10 10 Cell / mL, Bacteroides fragilis ATCC25285 bacterial suspension 1×10 10 Cell / mL.

[0042] Example 4: In vitro pharmacological effect of Bacteroides fragilis on gliadin-induced changes in Caco-2 cells. The main component of gluten is gliadin, which cannot be completely degraded by digestive enzymes in the gastrointestinal tract. This results in peptides rich in glutamine and proline, triggering both adaptive and innate immune responses in the gut, inducing apoptosis of intestinal epithelial cells, and disrupting tight junctions between cells, leading to intestinal barrier damage, increased intestinal permeability, and clinical symptoms such as chronic diarrhea and abdominal discomfort. This experiment used a Caco-2 cell model in vitro to investigate the effects of Bacteroides fragilis on gliadin-induced cell damage and decreased expression of tight junction proteins.

[0043] 4.1 Experimental Design and Procedure 4.1.1 Resuscitation and passage of Caco-2 cells Caco-2 cells were grown in DMEM medium (complete medium) containing 10% FBS, with 1% penicillin / streptomycin added, and cultured in an incubator at 37°C with 5% CO2 and saturated humidity, with the medium being changed every two days.

[0044] a) Remove the cell line from liquid nitrogen and thaw it rapidly in a 37°C water bath. Under aseptic conditions, open the cryovial, transfer the liquid to a 15mL centrifuge tube, resuspend the cells in 2-3mL of complete culture medium, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add 5mL of complete culture medium, and seed the cells in a culture flask. Incubate at 37°C in a 5% CO2 incubator for 48 h.

[0045] b) Discard the original culture medium, add 1 mL of 0.25% trypsin, digest for 5 min, add 3 mL of complete culture medium to stop the reaction, and transfer to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. Discard the supernatant, add appropriate complete culture medium, and inoculate the cells into culture flasks. Incubate at 37°C in a 5% CO2 incubator for 24 h.

[0046] 4.1.2 Effects of Bacteroides fragilis on transmembrane resistance and ZO-1 mRNA expression levels in Caco-2 monolayer cells (1) Establishment of Caco-2 monolayer cells Caco-2 cells were divided into 10 5 / cm 2 The cells were seeded at the appropriate density in 24-well Millicell chambers and cultured for 10-15 days. During this period, the transepithelial resistance (TER) of monolayer cells was measured using a Millicell ERS-2 transepithelial cell resistance meter (Millipore Corporate). The cells were cultured until the TER value reached 600 Ω / cm.2 If the cell fusion is successful, the monolayer is considered to be ready for testing.

[0047] (2) Experimental grouping Table 1 Experimental Groups and Drug Administration

[0048] (3) Experimental methods Before treatment with gliadin and live / inactivated bacteria, the confluent monolayers of cells were washed twice with PBS and incubated overnight in DMEM medium containing 10% FBS and 2 mM glutamine but no antibiotics.

[0049] ① Add the corresponding samples to the upper layer of the cell chamber according to the above grouping and incubate. Measure TER immediately after 6 hours of incubation. Make three replicates for each group.

[0050] ② Add the corresponding samples to the upper layer of the cell chamber according to the above grouping and incubate for 24 hours. Collect the cells and use qPCR to determine the mRNA expression level of tight junction protein ZO-1. Perform three replicates for each group.

[0051] (4) Testing items and methods Caco-2 cell transmembrane resistance: The integrity of the Caco-2 cell monolayer was assessed using transmembrane resistance (TEER) measured by an ERS meter. A TEER value greater than 600 Ω / cm was considered a successful cell fusion. A higher TEER value indicates greater cell integrity and a denser cell layer.

[0052] The expression level of tight junction protein ZO-1 mRNA was determined using qPCR and reflects intestinal barrier function.

[0053] Data statistics and analysis: SPSS 25.0 statistical software was used for statistical analysis.

[0054] 4.2 Experimental Results 4.2.1 Transmembrane resistance Table 2. Effect of Bacteroides fragilis on transmembrane resistance of Caco-2 cells (mean±SD, n=3)

[0055] Note: " "Indicates comparison with the model group, P <0.01; "Indicates comparison with the model group, P <0.001; ##"This indicates a comparison with the standard live bacteria group," P <0.01; ### "Indicates comparison with the model group, P <0.001; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01.

[0056] Table 2 shows the transmembrane resistance values ​​of cells before and after drug incubation. Compared with the blank control group, the transmembrane resistance value of the model group decreased after gliadin treatment, indicating cell damage. Compared with the model group, the transmembrane resistance of each ZY-312 treatment group was significantly increased, with statistical differences, while the transmembrane resistance of the ATCC25285 standard strain group was not statistically different from that of the model group. Compared with the standard live bacteria group, the transmembrane resistance of each ZY-312 live bacteria group was significantly increased, with statistical differences; compared with the standard inactivated bacteria group, the transmembrane resistance of each ZY-312 inactivated bacteria group was significantly increased, with statistical differences. The above results indicate that Bacteroides fragilis, especially Bacteroides fragilis ZY-312, can restore the transmembrane resistance value reduction induced by gliadin and has a cytoprotective effect.

[0057] 4.2.2 Expression level of tight junction protein ZO-1 mRNA Table 3. Effect of Bacteroides fragilis on ZO-1 mRNA expression levels (mean ± SD, n=3)

[0058] Note: " "Indicates comparison with the model group, P <0.01; "Indicates comparison with the model group, P <0.001; ## "This indicates a comparison with the standard live bacteria group," P <0.01; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01.

[0059] Table 3 shows the expression levels of the tight junction protein ZO-1 mRNA in Caco-2 cells by Bacteroides fragilis ZY-312. After treatment with gliadin (model group), ZO-1 expression in Caco-2 cells decreased significantly compared to the blank control group. After co-incubation of Caco-2 cells with gliadin and ZY-312 for 24 hours, ZO-1 mRNA expression was upregulated, showing a significant difference compared to the model group. However, the expression level of ZO-1 mRNA in the standard strain ATCC25285 showed no statistically significant difference compared to the model group. Compared to the live standard strain group, the expression of ZO-1 mRNA was upregulated in all live ZY-312 groups, with statistically significant differences. Compared to the inactivated standard strain group, the expression of ZO-1 mRNA was also upregulated in all inactivated ZY-312 groups, with statistically significant differences.

[0060] Both live and inactivated *Bacteroides fragilis* groups effectively improved the transmembrane resistance of intestinal cells treated with gliadin. Compared with the model group, the cell integrity was higher, indicating that both live and inactivated *Bacteroides fragilis* have cytoprotective effects. Both live and inactivated *Bacteroides fragilis* upregulated the expression of the tight protein ZO-1, reducing intestinal barrier damage caused by gliadin. These results suggest that *Bacteroides fragilis* ZY-312 has a good therapeutic effect on celiac disease.

[0061] Example 5: In vivo efficacy experiment of Bacteroides fragilis against gliadin-induced celiac disease. Gliadin-induced immune responses are very important in the pathogenesis of celiac disease, including increased anti-tissue transglutaminase (TTG) antibodies, increased expression of pro-inflammatory factors such as IFNγ and IL-15, leading to increased inflammation in intestinal tissues, causing intestinal epithelial apoptosis, pathologically manifested as villous atrophy and crypt hyperplasia; decreased expression of tight junction proteins, manifested as increased intestinal permeability.

[0062] 5.1 Experimental Design and Procedure 5.1.1 Establishment of animal models and experimental methods This experiment used an induced animal model, consisting of 64 newborn Wistar rats (half male and half female). Eight of them were normally raised, while the other 56 rats were given gavage with gliadin (5 mg / rat) combined with intraperitoneal injection of IFNγ (1000 U) on days 0, 3, and 20 after birth to establish a celiac disease rat model. The rats were then divided into groups, and the administration of drugs to each group began the following day. The specific grouping and administration methods are shown in Table 4, and subsequent experiments were conducted.

[0063] The rats were then divided into groups and administered gliadin once daily during the treatment period, 5 mg / rat / time.

[0064] 5.1.2 Experimental grouping and drug administration Table 4 Experimental Groups and Drug Administration

[0065] Rats were treated with gliadin via gavage combined with intraperitoneal injection of IFNγ on day 20 after birth, and then divided into groups. The next day, each group began receiving the treatment via gavage twice daily for two weeks, followed by subsequent experiments. 5.1.3 Detection Items and Methods Free glutamine and proline: After administration to rats, the rats were sacrificed, and 0.5 g of intestinal contents were collected. The intestinal contents were processed as follows: 1 g of the test sample was added to 5 mL of water, homogenized, and centrifuged at 10000 rpm for 5 min, then filtered. 1.5 mL of the filtrate was taken, and 0.5 mL of 10% trichloroacetic acid solution was added. The mixture was vortexed and centrifuged at 10000 rpm for 5 min. 500 μL of the supernatant was taken, and 500 μL of phenyl isothiocyanate derivatizing agent was added. The mixture was mixed and allowed to stand at room temperature for 30 min. 1 mL of n-hexane was added, the mixture was vortexed, and allowed to separate into layers. The lower layer was collected and filtered through a 0.22 μm filter membrane to prepare the sample. The free glutamine and proline contents of the sample were determined using high-performance liquid chromatography with a diode array detector (HPLC-DAD).

[0066] Small intestinal villus length: After sacrificing the rats, the duodenum was harvested to prepare HE sections, and the ratio of villus length to crypt depth was measured and calculated under a microscope of equal magnification.

[0067] Intestinal permeability assay: Intestinal permeability in mice was measured using fluorescein isothiocyanate-dextran (FITC-dextran 4000; Sigma). Mice were orally administered FITC-dextran (44 mg / 100 g) 4 hours before sacrifice. At the end of treatment, mice were anesthetized, and blood was collected via cardiac puncture into heparinized centrifuge tubes and centrifuged at 12000 × g for 10 min. Each plasma sample was diluted with an equal volume of pH 7.4 PBS, and a standard curve was constructed using serially diluted FITC-dextran stock solutions (0 ng / mL; 125 ng / mL; 250 ng / mL; 500 ng / mL; 1000 ng / mL; 2000 ng / mL; 4000 ng / mL; 8000 ng / mL). 100 μL of each diluted plasma was then transferred to a 96-well plate. Fluorescence was detected using a SPARK multimode microplate reader (485nm ex; 528nm em), and the FITC-glucan concentration of each sample was calculated using a standard curve.

[0068] Occludin mRNA expression level in small intestinal tissue: Occludin, as an important component of tight junctions, is very important for intestinal barrier permeability. Total RNA was extracted from mouse duodenum using the TRIzol method, and cDNA was obtained by reverse transcription using SuperScript II reverse transcriptase. The expression level of Occludin mRNA was detected by qPCR using cDNA as a template.

[0069] Serum anti-tissue transglutaminase (TTG) antibody: Serum-specific TTG-IgA antibody was detected using ELISA. The autoimmune response caused by TTG antibody is one of the factors leading to celiac disease.

[0070] Expression levels of small intestinal inflammatory factors IFNγ and IL-15 mRNA: Total RNA was extracted from mouse duodenum using the TRIzol method, and cDNA was obtained by reverse transcription using SuperScript II reverse transcriptase. The expression levels of IFNγ and IL-15 mRNA were detected by qPCR using cDNA as a template.

[0071] Data statistics and analysis: SPSS 25.0 statistical software was used for statistical analysis.

[0072] 5.2 Experimental Results 5.2.1 Content of free glutamine and proline in intestinal contents Table 5. Free glutamine and proline content (mean±SD, n=8)

[0073] Note: " "Indicates comparison with the model group, P <0.01; "Indicates comparison with the model group, P <0.001; ## "This indicates a comparison with the standard live bacteria group," P <0.01; ### "Indicates comparison with the model group, P <0.001; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01; +++ "This indicates a comparison with the standard strain of inactivated bacteria," P <0.001.

[0074] Glycol protein is degraded into indigestible peptides rich in glutamine and proline in the gastrointestinal tract, which is one of the factors contributing to celiac disease. In the normal control group, no glycol protein was administered. In the model group, after glycol protein administration, the levels of free glutamine and proline in the intestinal contents increased to some extent, indicating that some glycol protein can be digested and broken down into free amino acids. Compared with the model group, the levels of glutamine and proline in the intestinal contents of all ZY-312 treatment groups were increased (P < 0.001), indicating that ZY-312 helps break down indigestible peptides into absorbable free amino acids, reducing peptide irritation to the gastrointestinal tract and thus alleviating the severity of celiac disease. Compared with the standard live bacteria group, the levels of glutamine and proline in the intestinal contents of all ZY-312 live bacteria groups were increased, with statistically significant differences. Compared with the standard inactivated bacteria group, the levels of glutamine and proline in the intestinal contents of all ZY-312 inactivated bacteria groups were also increased, with statistically significant differences.

[0075] 5.2.2 Ratio of small intestinal villus length to crypt depth (V:C) Table 6. Results of detecting intestinal permeability of Bacteroides fragilis in rats (mean±SD, n=8)

[0076] Note:" "Indicates comparison with the model group, P <0.05; "Indicates comparison with the model group, P <0.01; "Indicates comparison with the model group, P <0.001; # "This indicates a comparison with the standard live bacteria group," P <0.05; ## "This indicates a comparison with the standard live bacteria group," P <0.01; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01.

[0077] Compared with the normal control group, the ratio of villus length to crypt depth in the model group was decreased, indicating shortened villus length and / or deepened crypts, consistent with the pathological manifestations of villus atrophy and crypt hyperplasia in celiac disease. Both live and inactivated Bacteroides fragilis improved the intestinal villus condition in rats, restoring the villus to normal length and the villus length-to-crypt depth ratio to normal. In particular, Bacteroides fragilis ZY-312 showed better potential for treating celiac disease. Compared with the standard live bacterium group, the ratio of villus length to crypt depth was increased in each ZY-312 live bacterium group, with statistically significant differences; the ratio was also increased in each ZY-312 inactivated bacterium group compared with the standard inactivated bacterium group, with statistically significant differences; indicating that ZY-312 restored intestinal villus better than the standard strain.

[0078] 5.2.3 Intestinal permeability testing Table 7. Results of Bacteroides fragilis detection of rat intestinal permeability (mean±SD, n=8)

[0079] Note: " "Indicates comparison with the model group, P <0.05; "Indicates comparison with the model group, P <0.001; # "This indicates a comparison with the standard live bacteria group," P <0.05; ## "This indicates a comparison with the standard live bacteria group," P <0.01; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01; +++ "This indicates a comparison with the standard strain of inactivated bacteria," P <0.001.

[0080] Higher plasma FITC-glucan concentrations indicate higher intestinal permeability and greater intestinal barrier damage in mice. Compared with the normally fed control group, the plasma FITC-glucan concentration in the model group was significantly increased, indicating that IFNγ+ gliadin disrupted the intestinal barrier in mice. After drug treatment, compared with the model group, all Bacteroides fragilis ZY-312 groups showed a decrease in plasma FITC-glucan concentration, with statistically significant differences. Both live and inactivated ZY-312 bacteria effectively improved IFNγ+ gliadin-induced increased intestinal barrier permeability. Compared with the standard live bacterial group, the plasma FITC-glucan concentration in each ZY-312 live bacterial group was lower, with statistically significant differences; compared with the standard inactivated bacterial group, the plasma FITC-glucan concentration in each ZY-312 inactivated bacterial group was lower, with statistically significant differences. This indicates that, compared with the standard strain, the live / inactivated strains of ZY-312 are more effective in improving IFNγ+ gliadin-induced intestinal barrier permeability.

[0081] 5.2.4 mRNA expression level of Occludin in small intestinal tissue Table 8. Effects of Bacteroides fragilis on the mRNA expression level of Occludin in small intestinal tissue (mean ± SD, n = 8)

[0082] Note: " "Indicates comparison with the model group, P <0.001; ## "This indicates a comparison with the standard live bacteria group," P <0.01; + "This indicates a comparison with the standard strain inactivated bacteria group," P <0.05; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01.

[0083] Occludin is one of the most representative proteins related to tight junctions, blocking cellular bypass pathways and forming the basic structure of tight junctions. Compared with the normal control group, the model group showed decreased Occludin mRNA expression. Compared with the model group, Occludin mRNA expression in all ZY-312 groups was significantly increased, showing statistical differences, while there was no statistically significant difference in Occludin mRNA expression between the ATCC25285 standard strain group and the model group. These results indicate that Bacteroides fragilis, especially Bacteroides fragilis ZY-312, can improve IFNγ-gliadin-induced Occludin expression decline and has a protective effect on the intestinal barrier. Compared with the live standard strain group, Occludin mRNA expression in all ZY-312 live strain groups was significantly increased, showing statistical differences; compared with the inactivated standard strain group, Occludin mRNA expression in all ZY-312 inactivated strain groups was significantly increased, showing statistical differences. This indicates that compared with the standard strain, the live / inactivated ZY-312 strains have a better effect on upregulating Occludin mRNA.

[0084] 5.2.5 Serum TTG-IgA antibody Table 9. Effects of Bacteroides fragilis on serum TTG antibody levels in celiac disease rats (mean ± SD, n = 8)

[0085] Note: " "Indicates comparison with the model group, P <0.01; "Indicates comparison with the model group, P <0.001; ## "This indicates a comparison with the standard live bacteria group," P <0.01; ### "Indicates comparison with the model group, P <0.001; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01; +++ "This indicates a comparison with the standard strain of inactivated bacteria," P <0.001.

[0086] The production of TTG-IgA antibodies leads to an autoimmune response, promoting celiac disease. Compared with the normal control group, the model group showed elevated serum TTG-IgA antibodies. Compared with the model group, the serum TTG-IgA antibody levels in all ZY-312 groups were significantly lower, with statistically significant differences. These results indicate that *Bacteroides fragilis*, especially *Bacteroides fragilis* ZY-312, can improve IFNγ-gliadin-induced elevation of serum TTG-IgA antibodies, exhibiting an immunomodulatory effect. Compared with the standard live bacterial group, the serum TTG-IgA antibody levels in all ZY-312 live bacterial groups were significantly lower, with statistically significant differences; compared with the standard inactivated bacterial group, the serum TTG-IgA antibody levels in all ZY-312 inactivated bacterial groups were significantly lower, with statistically significant differences. This suggests that compared with the standard strain, ZY-312's live / inactivated bacteria have a more significant effect in downregulating serum TTG-IgA antibodies.

[0087] 5.2.6 Expression levels of small intestinal inflammatory factors IFNγ and IL-15 mRNA Table 10. Effects of Bacteroides fragilis on the expression levels of inflammatory factors in the small intestine (mean ± SD, n = 8)

[0088] Note: " "Indicates comparison with the model group, P <0.01; "Indicates comparison with the model group, P <0.001; # "This indicates a comparison with the standard live bacteria group," P <0.05; ## "This indicates a comparison with the standard live bacteria group," P <0.01; + "This indicates a comparison with the standard strain inactivated bacteria group," P <0.05; ++ "This indicates a comparison with the standard strain inactivated bacteria group," P <0.01.

[0089] IFNγ and IL-15 are pro-inflammatory factors closely related to the pathogenesis of celiac disease. Compared with the blank control group, the serum IFNγ and IL-15 mRNA expression was increased in the model group. Compared with the model group, the IFNγ and IL-15 mRNA expression in all ZY-312 groups was significantly decreased, with statistically significant differences, while the IFNγ and IL-15 mRNA expression in the ATCC25285 standard strain group showed no statistically significant difference compared with the model group. These results indicate that each dose of live / inactivated ZY-312 can significantly improve the IFNγ-gliadin-induced increase in IFNγ and IL-15 mRNA expression, exhibiting an immunomodulatory effect. Compared with the standard live strain group, the ZY-312 live strain group significantly downregulated IFNγ and IL-15 mRNA expression, with statistically significant differences; compared with the standard inactivated strain group, the ZY-312 inactivated strain group significantly downregulated IFNγ and IL-15 mRNA expression, with statistically significant differences. This indicates that, compared with the standard strain, live / inactivated ZY-312 strains are more effective in improving the increase in IFNγ and IL-15 mRNA expression induced by IFNγ-gliadin.

[0090] The above results indicate that Bacteroides fragilis can improve IFNγ-glucosinolate-induced small intestinal villus atrophy, increase intestinal permeability, decrease the expression of tight junction protein Occludin, protect the intestinal barrier, reduce serum TTG-IgA antibody levels, and decrease the expression of small intestinal inflammatory factors IFNγ and IL-15, thus having immunomodulatory and anti-inflammatory effects and showing good potential in the treatment of celiac disease.

[0091] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of Bacteroides fragilis in the preparation of a medicament for treating celiac disease, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312 with accession number CGMCC No. 10685.

2. The application according to claim 1, characterized in that, The Bacteroides fragilis is either a live or inactivated bacterium; the inactivated bacterium is an inactivated bacterium with intact morphology and structure.

3. The application according to claim 1, characterized in that, The *Bacteroides fragilis* is either a live *Bacteroides fragilis* cell or an inactivated *Bacteroides fragilis* cell.

4. The application according to any one of claims 1-3, characterized in that, The celiac disease includes typical celiac disease, atypical celiac disease, or asymptomatic celiac disease.

5. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

6. The application according to claim 1, characterized in that, The dosage form of the drug is pills, tablets, powders, granules, capsules, solutions, or suspensions.

7. The application according to claim 1, characterized in that, The drug is in the form of tube feeding preparation, oral liquid or enema.

8. The use of a composition containing Bacteroides fragilis in the preparation of a medicament for treating celiac disease, wherein Bacteroides fragilis is Bacteroides fragilis ZY-312 with accession number CGMCC No. 10685.

9. The application according to claim 8, characterized in that, The drug also contains pharmaceutically acceptable carriers and / or excipients.

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