Lactobacillus reuteri and its use in the preparation of a medicament for the treatment of diffuse large b-cell lymphoma

Abstract

The application discloses Lactobacillus reuteri and application thereof in preparation of a medicine for treating diffuse large B-cell lymphoma, and relates to the technical field of biological medicine. The application provides application of Lactobacillus reuteri and / or a metabolite thereof in preparation of a medicine for treating diffuse large B-cell lymphoma, wherein the Lactobacillus reuteri is Lactobacillus reuteri (HG001). Limosilactobacillus reuteri The Lactobacillus reuteri is separated from feces of healthy people and used for preparation of the medicine for treating diffuse large B-cell lymphoma, and it is found that the Lactobacillus reuteri is confirmed as a probiotic strain for enhancing the chemotherapy effect of diffuse large B-cell lymphoma and can improve side effects caused by cyclophosphamide treatment of diffuse large B-cell lymphoma; and the Lactobacillus reuteri and the cyclophosphamide have a synergistic effect in treatment of diffuse large B-cell lymphoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a strain of *Lactobacillus reuteri* and its application in the preparation of a drug for treating diffuse large B-cell lymphoma. Background Technology

[0002] Non-Hodgkin lymphoma (NHL) is a highly heterogeneous malignant proliferative disease of lymphoid tissue and one of the most common hematologic malignancies. Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma in adults, accounting for approximately 30%-40% of all non-Hodgkin lymphomas. In China, over 60% of adult NHL cases are diagnosed as DLBCL. DLBCL exhibits high heterogeneity in morphology, biology, immunophenotypic characteristics, genetics, and clinical presentation. Currently, the preferred treatment regimen is multi-drug chemotherapy combined with rituximab (R-CHOP: rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Although treatment response rates have improved, one-third of patients still do not benefit from current treatment strategies and experience relapse and resistance. This is because the pathogenesis of DLBCL is not fully understood, limiting treatment options. In addition, the gastrointestinal side effects of chemotherapy drugs also lead to poor prognosis for many patients who cannot tolerate chemotherapy. Therefore, it is particularly important to find new treatment strategies to improve the clinical efficacy of DLBCL and reduce the severe gastrointestinal toxicity caused by chemotherapy drugs.

[0003] Cyclophosphamide (CTX) is a key drug in the treatment of DLBCL (drug-induced leukemia-associated lymphoid tissue disease). It is a nitrogen mustard derivative that exerts its effect primarily through hydrolysis by phosphoramidases or phosphatases present in the liver or tumor, converting it into the active form, phosphoramidite mustard. This drug has a broad antitumor spectrum and was the first so-called "latent" broad-spectrum antitumor drug, effective against both leukemia and solid tumors. Cyclophosphamide is inactive in vitro; its main function is exerted through hydrolysis by hepatic P450 enzymes into aldehyde phosphoramidite, which is then transported to tissues to form phosphoramidite mustard. Cyclophosphamide can be inactivated by dehydrogenases converting it to carboxyphosphamide, or excreted as acrolein, leading to urinary tract toxicity. It is a cell cycle nonspecific drug with a mechanism of action similar to nitrogen mustard. The main mechanism of action of cyclophosphamide is to inhibit DNA and protein synthesis in cancer cells, reducing cancer cell growth and spread. Inhibition of DNA synthesis is due to its unique chemical structure, which allows it to bind to phosphodiester bonds in DNA and prevent DNA replication. In addition, cyclophosphamide can also inhibit protein synthesis in cancer cells, thereby reducing the energy metabolism and material cycling of cancer cells by blocking the protein synthesis chain, and thus inhibiting the growth and spread of cancer cells.

[0004] However, cyclophosphamide (CTX) has significant toxic side effects. Major side effects associated with high doses or prolonged use of CTX include gastrointestinal side effects, hemorrhagic cystitis, nephrotoxicity, and cardiotoxicity. Many patients experience severe intestinal damage, including intestinal inflammation, intestinal barrier disruption, gut microbiota dysbiosis, and malabsorption, making them unable to tolerate subsequent chemotherapy drugs and increasing the risk of cancer-related cachexia. This leads to decreased patient compliance and chemotherapy tolerance, resulting in poorer prognosis. To improve this situation, protective agents are increasingly used to mitigate adverse side effects in chemotherapy patients; among these, the use of gut microbiota modulation to alleviate CTX-induced immunosuppression is receiving increasing attention. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a strain of *Lactobacillus reuteri* and its application in the preparation of a drug for treating diffuse large B-cell lymphoma.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a strain of *Lactobacillus reuteri*, wherein the *Lactobacillus reuteri* is *Lactobacillus reuteri* (… Limosilactobacillus reuteri HG001, Lactobacillus reuteri HG001, was deposited on October 20, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.36248.

[0008] Secondly, the present invention provides the use of the aforementioned *Lactobacillus reuteri* and / or its metabolites in the preparation of a medicament for treating diffuse large B-cell lymphoma.

[0009] Optionally, the application includes preparing a drug that improves the side effects of cyclophosphamide in the treatment of diffuse large B-cell lymphoma.

[0010] Optionally, the drug reduces the gastrointestinal side effects of cyclophosphamide treatment for diffuse large B-cell lymphoma by at least one of the following methods:

[0011] (1) Enhance the α- and β-diversity of gut microbiota;

[0012] (2) Increase the abundance of Lactobacillus and Firmicutes in the gut;

[0013] (3) Relieves intestinal microenvironment disorder and barrier damage caused by chemotherapy.

[0014] Optionally, the metabolites of *Lactobacillus reuteri* are at least one of indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid, wherein indole-3-carboxaldehyde is produced by *Lactobacillus reuteri* through tryptophan metabolism.

[0015] Thirdly, the present invention provides the use of the aforementioned *Lactobacillus reuteri* and / or its metabolites in synergistic use with cyclophosphamide in the preparation of a pharmaceutical combination for the treatment of diffuse large B-cell lymphoma.

[0016] Optionally, the drug combination inhibits tumor growth through at least one of the following mechanisms:

[0017] (1) The tumor volume and weight decreased;

[0018] (2) Slowed tumor growth;

[0019] (3) Decreased levels of serum markers related to tumor burden, including LDH, SF, and β2-MG.

[0020] Optionally, the metabolite of *Lactobacillus reuteri* is at least one of indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid, wherein the indole-3-carboxaldehyde is produced by *Lactobacillus reuteri* through tryptophan metabolism;

[0021] The *Lactobacillus reuteri* and its metabolite indole-3-carboxaldehyde can enhance the chemosensitivity of cyclophosphamide to diffuse large B-cell lymphoma by activating aryl hydrocarbon receptors.

[0022] Thirdly, the present invention provides a medicament for treating diffuse large B-cell lymphoma, the medicament comprising the aforementioned *Lactobacillus reuteri* and / or its metabolites.

[0023] Optionally, the drug also includes cyclophosphamide.

[0024] This invention has at least one of the following beneficial effects:

[0025] 1. This invention isolates a strain of *Lactobacillus reuteri* from the feces of healthy individuals, evaluates its probiotic properties, and further assesses its in vivo antitumor activity. The strain is then used to prepare a drug for treating diffuse large B-cell lymphoma. *Lactobacillus reuteri* has been identified as a probiotic strain that enhances the efficacy of chemotherapy for diffuse large B-cell lymphoma. It can improve the side effects caused by cyclophosphamide treatment for diffuse large B-cell lymphoma, including improving gastrointestinal side effects. The α and β diversity of the gut microbiota are higher than in the MC group, and the abundance of *Lactobacillus* and *Firmwallis* is increased. The abundance of *Lactobacillus* is negatively correlated with inflammation and tumor burden.

[0026] 2. This invention also found that *Lactobacillus reuteri* and cyclophosphamide have a synergistic effect in the treatment of diffuse large B-cell lymphoma. Experiments showed that compared with cyclophosphamide chemotherapy alone (MC group), the combination with *Lactobacillus reuteri* (MCL group) can more significantly inhibit tumor growth, manifested as a reduction in tumor volume and weight, a delay in tumor growth, and a decrease in the levels of serum markers related to tumor burden such as LDH, SF, and β2-MG.

[0027] 3. This invention further discovers that three metabolites of *Lactobacillus reuteri*—indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid—all have inhibitory effects on DLBCL cell proliferation. Furthermore, the inhibition of DLBCL cells by these three metabolites exhibits a time- and concentration-dependent trend, with indole-3-carboxaldehyde showing the most significant inhibitory effect and xanthuric acid showing the weakest. Moreover, this invention also confirms that indole-3-carboxaldehyde, produced by *Lactobacillus reuteri* through tryptophan metabolism, can inhibit DLBCL, and tryptophan deficiency can reverse this effect and exacerbate barrier damage. Attached Figure Description

[0028] Figure 1 The results for Example 2 are shown below. In this table, A represents the change in mouse body weight, B represents the change in tumor volume, C represents the change in tumor weight, D represents the lactate dehydrogenase content, E represents the β2-microglobulin content, F represents the serum ferritin content, G represents the serum IL-1β content, H represents the serum TNF-α content, I represents the serum IL-6 content, J represents the linear discriminant analysis (LDA) distribution histogram of differential species, K represents the α diversity of microbial composition in mouse feces after the intervention, and L represents the β diversity of microbial composition in mouse feces after the intervention.

[0029] Figure 2 The results of Example 3 are shown below. In Example 3, A represents the inhibitory efficiency of live *Lactobacillus reuteri* diluted in different proportions and co-incubated with tumor cells on tumor cells; B represents the inhibitory efficiency of heat-inactivated *Lactobacillus reuteri* diluted in different proportions and co-incubated with tumor cells on tumor cells; C represents the inhibitory efficiency of *Lactobacillus reuteri* supernatant diluted in different proportions and co-incubated with tumor cells on tumor cells; D represents the Western Blot analysis of the apoptosis-related protein levels of tumor cells after co-intervention with chemotherapy drugs using different components of *Lactobacillus reuteri* (live bacteria, supernatant, and inactivated bacteria); E represents the Caspase3 protein quantification bar chart; F represents the Cleaved-Caspase3 protein quantification bar chart; G represents the Bcl-2 protein quantification bar chart; and H represents the Bax protein quantification bar chart.

[0030] Figure 3The results of Example 3 are shown below. A represents a heatmap of species composition from MRS medium containing *Lactobacillus reuteri* supernatant, analyzed using tryptophan-targeted metabolomics. B represents β-diversity in targeted metabolomics. C represents the relative abundance of indole-3-lactic acid in targeted tryptophan metabolomics analysis. D represents the relative abundance of indole-3-carboxaldehyde. E represents the relative abundance of xanthurenic acid. F represents the 48-hour inhibition rate of the three metabolites (indole-3-lactic acid, indole-3-carboxaldehyde, and xanthurenic acid) on SU-DHL-4 cell proliferation compared to PBS. G represents the 24-hour inhibition rate of the three metabolites (indole-3-lactic acid, indole-3-carboxaldehyde, and xanthurenic acid) on OCI-LY3 cell proliferation compared to PBS. H represents the 24-hour inhibition rate of the three metabolites (indole-3-lactic acid, indole-3-carboxaldehyde, and xanthuric acid) on SU-DHL-4 cell proliferation compared to PBS; I represents the 48-hour inhibition rate of the three metabolites (indole-3-lactic acid, indole-3-carboxaldehyde, and xanthuric acid) on OCI-LY3 cell proliferation compared to PBS; J represents the 24-hour inhibition rate curves of the three metabolites (indole-3-lactic acid, indole-3-carboxaldehyde, and xanthuric acid) on OCI-LY3 cell proliferation at different concentrations; and K represents the 24-hour inhibition rate curves of the three metabolites (indole-3-lactic acid, indole-3-carboxaldehyde, and xanthuric acid) on SU-DHL-4 cell proliferation at different concentrations.

[0031] Figure 4 The results of Example 4 are shown below. In this chart, A represents the change in mouse body weight, B represents the change in tumor volume, C represents the tumor weight of each group, D represents the β2-microglobulin content of each group, E represents the lactate dehydrogenase content of each group, F represents the tumor weight and volume bar chart of each group, G represents the serum ferritin content of each group, H represents the indole-3-carboxaldehyde content of each group, I represents the proportion of Ki67 positive cells in the tumor tissue of each group, J represents the colon length bar chart of each group, and K represents the representative immunohistochemical staining of Ki67 in the tumor tissue of each group after intervention.

[0032] Figure 5 The results of Example 4 are shown below. In this table, A represents the change in tumor volume, B represents the change in mouse body weight, C represents the tumor weight of each group, D represents the tumor volume of each group, E represents the β2-microglobulin content, serum ferritin content, and lactate dehydrogenase content of each group, F represents the colon length of each group, G represents the indole-3-carboxaldehyde of each group, H represents a schematic diagram of the colon length of each group and a representative image of immunohistochemical staining for ZO-1 and P53 indicators, I represents the relative fluorescence intensity of ZO-1 in each group, and J represents the proportion of P53 positive cells in each group.

[0033] Figure 6The results of Example 4 are shown below. In this table, A represents the change in mouse body weight, B represents the change in tumor volume, C represents the tumor volume of each group, D represents the serum ferritin content of each group, E represents the β2-microglobulin content of each group, F represents the lactate dehydrogenase content of each group, G represents the proportion of K67 positive cells in each group, H represents the β2-microglobulin content, tumor weight, tumor volume, serum ferritin, proportion of P53 positive cells, proportion of K67 positive cells, and TNF-α content, I represents the serum IL-10 content, and I represents a representative image of P53 and Ki67 immunohistochemical staining in tumor tissue after the intervention.

[0034] Figure 7 The results of the identification of the probiotic properties of *Lactobacillus reuteri* in Example 1 are shown below. (A) Electron micrograph of *Lactobacillus reuteri* (scale bar = 5 μm). (B) Identification of the antioxidant properties of *Lactobacillus reuteri*. (C) Growth curve of *Lactobacillus reuteri*. (D) Schematic diagram of bile salt tolerance test. (E) Schematic diagram of acid tolerance test. (F) Bar graph of inhibition zone diameter in acid tolerance test (n=3). (G) Schematic diagram of inhibition test. (H) Bar graph of inhibition zone diameter in inhibition test (n=3). (I) Resistance test using the paper disc diffusion method for *Lactobacillus reuteri*; C: chloramphenicol (30 μg tablet), CTR: ceftriaxone (30 μg tablet), TET: tetracycline (30 μg tablet), GEN: gentamicin (10 μg tablet), PEN: penicillin (10 μg tablet), AMP: ampicillin (10 μg tablet), MY: lincomycin (2 μg / tablet), CIP: ciprofloxacin (5 μg / tablet), SXT: trimethoprim-sulfamethoxazole (25 μg / tablet), E: erythromycin (15 μg / tablet). (J) Bar chart of inhibition zone diameter in the resistance test (n=3).

[0035] Note: In the above figures, "Lactobacillus reuteri" and "Lactobacillus reuteri" both refer to the Lactobacillus reuteri of the present invention. Limosilactobacillus reuteri HG001. Detailed Implementation

[0036] Example 1: Screening, Identification, and In Vitro Probiotic Experiments of Strains

[0037] I. Screening: In this invention, *Lactobacillus reuteri* was isolated from normal healthy human fecal samples. The specific isolation method is as follows: Fecal samples from healthy individuals (must be in suspension) were added to sterile physiological saline or PBS and homogenized. Then, they were centrifuged at low speed (1000×g, 5 min) to remove large particles. The supernatant was collected (or large particulate impurities were removed using a 0.45 μm filter membrane), diluted to different proportions, and spread onto MRS solid medium. The samples were cultured for 24 hours in both aerobic and anaerobic environments. Single bacteria were selected and cultured further in MRS liquid medium for 24 hours. Strains with good growth were selected for DNA extraction, identification, and preservation, and designated as HG001.

[0038] II. Identification: Through comprehensive analysis of the strain's morphology, physiological and biochemical characteristics, 16S rRNA gene sequence (as shown in SEQ ID No. 1), and dnaK gene sequence, strain HG001 was ultimately identified as *Lactobacillus reuteri* (…). Limosilactobacillus reuteri The strain was deposited on October 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.36248.

[0039] III. In vitro probiotic experiments:

[0040] (1) Growth curve: After activating the probiotics in MRS medium, the initial activation was completed by overnight incubation at 37℃ (aerobic conditions). The next day, 1 mL of bacterial culture was inoculated into 200 mL of fresh medium and placed in a shaker or anaerobic jar at 37℃ for incubation. The optical density of the culture medium was measured periodically using a spectrophotometer (OD600 nm) and recorded every 2 hours to continuously monitor the growth changes of bacteria over 24-48 hours. At the same time, serial dilutions were performed and plate counts were performed to calculate the actual number of viable bacteria.

[0041] (2) Acid tolerance test: After activating the probiotics in MRS medium, they were cultured overnight at 37°C (aerobic) to complete the initial activation. The next day, the same medium was used for re-inoculation (the second generation bacteria had better activity), and they were cultured at 37°C for 24-48 hours. 1 mL of bacterial culture was taken out, centrifuged (6000 rpm for 4 minutes) to remove the supernatant, and the remaining bacteria were washed with PBS to remove the medium. The bacteria were then resuspended in phosphate buffered saline (PBS) at different pH values ​​(3.0, 4.0, 5.0, 6.0) and incubated at 37°C for 4 hours. Subsequently, the number of viable bacteria under each pH condition was calculated by plate counting.

[0042] (3) Bile salt tolerance test: After activating probiotics in MRS medium, the bacteria were cultured overnight at 37°C (aerobic) to complete the initial activation. The next day, the same medium was used for re-inoculation (second-generation bacteria showed better activity), and the bacteria were cultured at 37°C for 24-48 hours. 1 mL of bacterial culture was taken out, centrifuged (6000 rpm for 4 minutes) to remove the supernatant, and the remaining bacteria were washed with PBS to remove the medium. The bacteria were then resuspended in phosphate buffer (PBS) with different bile salt concentrations (0%, 0.1%, 0.2%, 0.3%) and incubated at 37°C for 4 hours. Subsequently, the viable bacteria count under each condition was calculated by plate counting.

[0043] (4) Antibacterial experiment: Common foodborne pathogens such as Salmonella rodentata, Candida albicans, Salmonella enteritidis, hemolytic streptococci, Escherichia coli, Shigella flexneri, Shigella dysenteriae, and Staphylococcus aureus were cultured in soybean broth (LB) liquid medium. After 24 hours of culture, the bacterial solution was evenly spread on the surface of LB agar medium. Then, Oxford cups were placed, and 250 μL of culture supernatant of the cultured probiotics was added to them. The control group used MRS medium. After 6-8 hours of observation, the size of the inhibition zone of pathogen growth was measured.

[0044] (5) Antibiotic resistance test: Take 30 μL of activated probiotic bacterial solution and spread it evenly on the surface of agar plates. Then place antimicrobial susceptibility test discs (chloramphenicol (30 μg tablets), ceftriaxone (30 μg tablets), tetracycline (30 μg tablets), gentamicin (10 μg tablets), penicillin (10 μg tablets), ampicillin (10 μg tablets), lincomycin (2 μg / tablet), ciprofloxacin (5 μg / tablet), compound sulfamethoxazole (25 μg / tablet), erythromycin (15 μg / tablet)) on the agar surface. Incubate the plates in a 37℃ incubator for 16 to 18 hours. After incubation, measure and record the diameter of the inhibition zone, calculate its MIC value, and refer to the breakpoint value of the corresponding species in the "General Rules for Probiotics for Food" to determine the antibiotic resistance results of the strains.

[0045] (6) Antioxidant experiment:

[0046] ① DPPH free radical scavenging capacity determination: In the experimental group, 2 mL of culture supernatant was added to 2 mL of 0.2 mmol / L DPPH methanol solution, and the reaction was carried out at room temperature in the dark for 30 minutes. After the reaction was completed, the absorbance of the solution at a wavelength of 517 nm (OD517 nm) was measured using a spectrophotometer. In the control group, an equal volume of culture medium was used instead of the sample, and the remaining steps were the same as those in the experimental group.

[0047] A0 blank group = 2 mL culture medium + 2 mL DPPH methanol solution; A1 experimental group = 2 mL culture supernatant + 2 mL DPPH methanol solution; A2 control group = 2 mL culture supernatant + 2 mL deionized water;

[0048] The formula for calculating the DPPH radical scavenging rate is: DPPH radical scavenging rate = [(A0 - (A1 - A2)) / A0] × 100%;

[0049] ② Determination of hydroxyl radical (-OH) scavenging ability: In a test tube, add 1 mL of 2 mmol / L FeSO4 solution, 1 mL of 6 mmol / L H2O2, and 1 mL of bacterial culture supernatant sequentially. Let stand for 10 min, then add 2 mL of 6 mmol / L salicylic acid. Under room temperature conditions, let stand for 30 min, and measure the absorbance at 510 nm to calculate the hydroxyl radical scavenging rate.

[0050] A0 blank group = 1 mL FeSO4 solution + 1 mL H2O2 + 1 mL culture medium + 2 mL salicylic acid;

[0051] Experimental group A1 = 1 mL FeSO4 solution + 1 mL H2O2 + 1 mL culture supernatant + 2 mL salicylic acid;

[0052] Hydroxyl radical scavenging rate = [(A0-A1) / A0] × 100%;

[0053] ③ Superoxide radicals (O2) - Scavenging capacity assay: Add 2 mL of 150 mmol / L Tris-HCl solution (pH=8.0) and 1 mL of 1.2 mmol / L pyrogallol solution sequentially to 0.5 mL of culture supernatant, shake rapidly, and measure the absorbance at 330 nm every 30 s until 150 s. Calculate the rate.

[0054] A0: The self-oxidation rate is obtained by fitting a straight line to the absorbance value of pyrogallol every 30 seconds and taking its slope; A1: The oxidation rate is obtained by fitting a straight line to the absorbance value of the mixture of pyrogallol and bacterial supernatant every 30 seconds and taking its slope.

[0055] Superoxide radical scavenging rate = (A0 - A1) / A0 × 100%

[0056] ④ Ferrous ions (Fe 2+Determination of chelating ability: Take 0.5 mL of bacterial culture, add 0.1 mL of 0.4% FeSO4 solution, mix, then add 0.1 mL of 1% vitamin C solution and 1 mL of 0.2 mol / L NaOH solution, and react at room temperature for 20 minutes. Then add 1 mL of 10% trichloroacetic acid solution, and centrifuge at 6000 rpm for 10 minutes at 4℃ to remove protein. Take 0.4 mL of the supernatant, add 4 mL of 0.1% o-phenanthroline solution, and react at room temperature for 10 minutes. Measure the absorbance (OD) of the solution at 546 nm using a spectrophotometer. 546 nm The formula for calculating the chelating capacity of ferrous ions is:

[0057] A0 blank group: absorbance after replacing the bacterial culture medium in the reaction system with 0.5 mL of culture medium; A1 experimental group: absorbance after reacting with 0.5 mL of sample solution; A2 control group: absorbance after replacing the ferrous sulfate solution in the reaction system with 0.1 mL of deionized water.

[0058] Fe 2+ Chelating capacity = [A0 - (A1 - A2)] / A0 × 100%;

[0059] ⑤ Total reducing power determination: Take 1 mL of bacterial culture, add 1 mL of PBS (pH = 6.6) and 1 mL of 1% K3[Fe(CN)6] solution, mix, and react at 50 ℃ for 2 minutes. Add 1 mL of 10% trichloroacetic acid solution, mix well, take 1 mL of the mixture, add 4 mL of deionized water and 0.4 mL of 0.1% FeCl3 solution, and let stand for 10 minutes. Use deionized water as a blank control group. Measure the absorbance of the solution at 700 nm wavelength (OD700 nm) using a spectrophotometer. The intensity of reducing power is directly proportional to the absorbance of the reaction solution; the higher the absorbance, the stronger the reducing power of the analyte.

[0060] A1: All samples in the experimental group were added; A0: 1 mL of culture medium was used to replace the samples in the control group, and all other samples were added.

[0061] Total reducing force = A1 - A0.

[0062] The results are as follows Figure 7 As shown in the figure, this strain exhibits good in vitro probiotic properties.

[0063] Example 2

[0064] To verify that Lactobacillus reuteri HG001 improves the gastrointestinal toxicity of cyclophosphamide treatment for DLBCL and enhances the efficacy of the treatment, the following animal experiments were conducted.

[0065] I. Experimental Preparation:

[0066] (1) Cell line (SUDHL-4): SUDHL-4 is a human GCB-like DLBCL cell line. The initial density was 2.5 x 10⁻⁶ cells. 5 Cells were placed in T25 cell culture flasks containing RPMI 1640 medium with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, and 30 μg / ml glutamine, and cultured in an incubator at 37°C, 5% CO2, and saturated humidity. After the first passage in 2-3 days, the cells were transferred to T75 cell culture flasks, and subsequently transferred to T150 cell culture flasks as needed based on cell growth. The SU-DHL-4 cell line used in the experiment was the same cell line purchased from the Typical Culture Cell Bank of the Chinese Academy of Sciences.

[0067] (2) Experimental animals: Before conducting in vivo animal experiments, this study obtained the consent of the school's ethics committee through the application and approval procedures. All experimental animal operations were carried out in accordance with relevant ethical regulations. 5-6 week old female NSG mice were housed in an SPF-grade experimental animal room and kept under a cycle of 12 hours of light and 12 hours of darkness. There were 6 mice per cage and 2 cages per group. The mice had free access to food and water. Before the start of the experiment, the mice were given a 5-day acclimatization feeding period. The temperature, humidity, noise and other conditions of the animal room were controlled within the specified range and cleaned regularly every day.

[0068] (3) DLBCL tumor-bearing model: DLBCL cells in the logarithmic growth phase and in good growth condition were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded; the cells were resuspended in sterile PBS solution and counted under a microscope; sterile PBS solution was added to readjust the cell density to 1*10 8 Cells / mL, stored on ice; add an equal volume of Matrigel and mix well; the cell density at this point is 0.5*10⁻⁶. 8 Cells / mL; 200 μL of cell suspension was inoculated subcutaneously into the outer side of the right hind limb of the mouse. Oval wheals were visible to the naked eye. The mice were placed in a sterile environment for further culture. The general condition, weight and tumor formation of the mice were observed daily.

[0069] (4) Cyclophosphamide administration strategy: Cyclophosphamide was administered intraperitoneally at a dose of 30 mg / kg, once every other day for 5 consecutive times. Survival time was assessed, and mice were sacrificed after the experiment. Tumor formation, spleen (spleen index), liver (liver index), and ileocolon were analyzed.

[0070] (5) Transplantation strategy of *Lactobacillus reuteri* and its metabolites: Based on the characteristics of the bacteria, select a suitable culture medium and culture the bacteria under specific temperature and culture conditions. Frozen bacterial culture samples are thawed at 37.5℃ for 10 minutes before use; lyophilized bacterial powder is added to the corresponding culture medium and thawed at 37.5℃ for 10 minutes before use. Generally, bacteria reach the logarithmic growth phase 8-10 hours after culture. Use a pipette to aspirate 1 mL of bacterial culture from the logarithmic growth phase culture into the spectrophotometer sample cell, and use a UV spectrophotometer to detect the OD600 value (Optical Density) to determine the bacterial concentration. When OD600≈1, the bacterial concentration is approximately 10... 8 -10 9 CFU / mL. After centrifuging the bacterial stock solution at 6000 rpm for 5 minutes, the supernatant was removed, and the precipitate was resuspended in sterile PBS to prepare approximately 10... 8 -10 9 CFU / mL bacterial suspension. Aliquot the diluted bacterial suspension into sterile centrifuge tubes and seal them. Transfer the sample to the isolator according to aseptic transfer requirements and administer 100 μL (1*10) to the animals via gavage. 9 (CFU / mL), once every other day for 4 consecutive weeks.

[0071] II. Experimental Grouping and Detection Indicators:

[0072] 1. Experimental grouping: 5-6 week old NSG female mice were selected and randomly divided into 3 groups of 12 mice each. The mice were housed in cages of 6 and fed with appropriate diet.

[0073] Group M: Model (DLBCL);

[0074] MC group: Model + cyclophosphamide (DLBCL + CTX);

[0075] MCL group: Model + Cyclophosphamide + Lactobacillus reuteri HG001 (DLBCL + CTX + bacteria).

[0076] 2. Detection indicators:

[0077] 2.1 General Conditions of Experimental Animals: During the experiment, the weight of mice was measured every two days. Measurements were taken in the early morning by slowly placing the mice on an electronic balance and reading the value after stabilization. Three stable values ​​were recorded consecutively, and the average value was taken as the final weight. The coat color, mental state, activity, food and water intake, defecation, limb swelling, paralysis, and survival of mice in each group were observed. Survival curves were plotted after the experiment. Every two days, the long and short diameters of the tumor were measured using calipers, and the tumor volume was calculated to plot a tumor growth curve. V (mm) 3 ) = (major axis L × minor axis W) 2 ) / 2.

[0078] 2.2 Feces: (1) Fecal sampling method: Fix the mouse with one hand, disinfect the skin near the anus with an alcohol swab, and induce the mouse to defecate by rectal massage. Collect fecal particles directly at the anus using a sterile tissue cryopreservation tube (take 2-3 fecal particles each time). The cryopreservation tube containing fecal particles should be immediately placed in liquid nitrogen for quick freezing and transferred to a -80℃ freezer for storage as soon as possible. During the whole process, care should be taken to avoid contact between the tube opening and the anal skin to avoid contamination by other bacteria. (2) 16S rRNA sequencing analysis of the differences in intestinal flora among the groups of mice: High-throughput sequencing: 1) Extraction and PCR amplification of genomic DNA: Genomic DNA was extracted from fecal samples using cetyltrimethylammonium bromide (CTAB method), and the purity and concentration of DNA were determined. The sample was diluted with sterile water to 1 ng / u L. 1) Using diluted genomic DNA as a template, polymerase chain reaction (PCR) amplification was performed using primer sequences for the V3 and V4 regions of 16S rRNA; 2) Mixing and purification of PCR products: PCR products were detected by electrophoresis using a 2% agarose gel; equal volumes of PCR products were mixed thoroughly and then detected by electrophoresis, and the target band was recovered using a gel extraction kit; 3) Library construction and sequencing: The TruSeq⑧ DNAPCR-Free Sample Preparation Kit was used for library construction. The constructed library was quantified by Qubit and q-PCR. After the library was qualified, it was sequenced using a HiSeq2500PE250.

[0079] Sequencing data processing: Based on specific sequences and PCR amplification primer sequences, sample data are separated from the sequencing data. Specific sequences and primer sequences are removed, and reads from each sample are assembled using FLASH. The resulting assembled sequences are the raw tags. The assembled raw tags undergo rigorous filtering to obtain high-quality clean tags. Chimeric sequences are removed from the clean tags to obtain the final effective tags.

[0080] OTU Clustering and Species Annotation: All effective tags of all samples were clustered using Uparse software. By default, sequences were clustered into OTUs (Operational Taxonomic Units) with 97% identity. Species annotation was performed on the representative sequences of OTUs. Species annotation analysis was performed using the Mothur method and the SILVA SSUrRNA database (with a threshold of 0.8-1). Community composition of each sample was statistically analyzed at each taxonomic level: kingdom, phylum, class, order, family, genus, and species.

[0081] Analysis of Alpha diversity, Beta diversity, and species abundance: 1) Alpha diversity analysis: (a) Dilution curve plotting using R: The dilution curve describes the diversity of a sample, directly reflecting the reasonableness of the amount of sequencing data related to the sample, and indirectly reflecting the richness of species in the sample; (b) Calculating the Shannon index using Qiime software: This is a commonly used index for Alpha diversity analysis of bacterial communities, reflecting the difference in species diversity and homogeneity between different samples. The larger the Shannon index, the higher the bacterial community diversity. 2) Beta diversity analysis: This compares and analyzes the bacterial community composition between two groups of samples. Principal coordinate analysis (PCoA) is a commonly used Beta diversity analysis method, which is based on the unweighted unifrac distance. The greater the distance between samples, the greater the difference in diversity between samples. 3) Species abundance analysis: T-test and LEfSe analysis are used. LEfSe analysis is a software analysis that can find biomarkers with statistically significant differences between groups.

[0082] 2.3 Methods for obtaining mouse tissue samples: Mice in each group were fasted and deprived of water for 12 hours, then anesthetized with isoflurane inhalation and euthanized. Blood was collected from the eyeballs of mice in each group. After coagulation at room temperature for 1 hour, the blood was centrifuged at 3500 r / min for 15 min, and the supernatant was collected and stored at -80℃ for later use. After blood collection, mice were euthanized by cervical dislocation. The limbs and tails of the mice were fixed with medical tape. A midline incision was made in the abdomen, and the skin, subcutaneous tissue, and muscle were sequentially incised along the linea alba. The peritoneum was opened to enter the abdominal cavity, and the abdominal incision was opened with a retractor. Two cm of jejunum, ileum, and colon tissue were collected, washed with sterile PBS, and fixed in 4% paraformaldehyde. A 10 cm intestinal segment was immersed in sterile KHBB buffer for intestinal mucosal permeability testing. A portion of the intestinal segment was weighed, washed with sterile PBS, and quickly frozen at -80℃ for the determination of inflammatory factors.

[0083] 2.4 Ileum: (1) Detection of ileal mucosal permeability: Take 10 cm of fresh ileal tissue from KHBB buffer (pH 7.4), tie one end with a ligature, inject 100 μL of FITC-dextran4 (FD-4) (FITC-labeled dextran, 4 kDa) solution into the other end of the gavage needle, and then tie the other end to form an 8 cm intestinal tube. Place it in 2 mL of KHBB liquid and incubate at 37℃ for 20 min. Measure the FD-4 solution permeating into the incubation buffer in the intestinal tube by fluorescence spectrophotometry with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Measure the amount of FD-4 permeating per centimeter of small intestine per minute. (2) ELISA determination of inflammatory factors (IL-6, TNF-α, IL-10, etc.) in mouse ileal tissue: Accurately weigh mouse ileal tissue, add phosphate buffer at a ratio of 1:9 (m / V), and homogenize in a tissue homogenizer. Centrifuge at 3000 r / min for 10 min at 4℃ using a high-speed refrigerated centrifuge, collect the supernatant liquid after centrifugation and preserve it, which is the ileal tissue homogenate sample.

[0084] Remove the test samples and kit from the refrigerator and let them stand for 30 minutes to allow them to reach room temperature. Once room temperature is reached, shake the test solution well and strictly follow the instructions: a) Prepare distilled water and dilute the concentrated wash solution 20 times; prepare multiple concentrations of standards before the formal experiment and plot a standard curve: take 1 ml of the prepared standard and reconstitute the lyophilized standard (la) to achieve a concentration of 2000 pg / ml, shake well, and then dilute at decreasing ratios of 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 0 pg / ml. 15 minutes before formal use, prepare the working solution by mixing the concentrated biotinylated antibody (2a) and the biotinylated antibody diluent (2b) at a ratio of 200:1, and mixing the diluted concentrated enzyme conjugate (3a) and the enzyme conjugate diluent (3b) at a ratio of 200:1. a) Take the test sample and the strips from the kit that have been placed at room temperature. Seal and store any unused strips. b) Add the specimen and standard to different wells. Seal the reaction wells with sealing tape and then incubate them in an incubator at room temperature for 2 hours. During this period, it is necessary to manually mix it multiple times, with an interval of 10 minutes; c) Washing: After cleaning the liquid in the wells, add washing solution to each well, let it stand for 30 minutes, then shake off the washing solution, and aspirate the water in the wells 4 times; d) Add biotinylated antibody solution to the reaction wells, the amount of which is 100 μL in each reaction well. After adding, seal the reaction wells and then incubate them at room temperature for 2 hours; e) Washing: After cleaning the liquid in the wells, add washing solution to each well, let it stand for 30 minutes, then shake off the washing solution, and aspirate the water in the wells 4 times; f) Add enzyme conjugate to the reaction wells, the amount of which is 100 μL in each reaction well. After adding, seal the reaction wells, mix thoroughly, and then incubate them at room temperature for 0.5 hours. After cleaning the liquid from the wells, add washing solution to each well, let stand for 30 minutes, then shake off the washing solution and aspirate the water from the wells four times; g) Add 100 μL of colorimetric reagent to each reaction well, seal the wells, mix thoroughly, and incubate at room temperature in an incubator for 10-30 minutes in the dark, depending on the color intensity after 0.5 hours of incubation; h) Add 100 μL of stop solution to each reaction well, mix thoroughly, and then measure the OD450 value using a microplate reader for no more than 5 minutes; i) Smoothly link the measured OD450 value with the standard concentration, and obtain the corresponding concentration from the sample OD value;

[0085] (3) Western blot was used to detect the levels of tight junction proteins (ZO-1, claudin-1) in the ileum of mice.

[0086] SDS-PAGE gel electrophoresis: a) Prepare a 10% separating gel using a 50ml centrifuge tube. After adding the reagents, shake the centrifuge tube to mix thoroughly. Transfer the separating gel to a sandwich glass plate, leaving space approximately the same width as the comb for adding the stacking gel. Observe whether the glass plate is completely sealed. Add isopropanol dropwise onto the separating gel and completely cover it until the gel aggregates. Gently discard the isopropanol on top of the separating gel and blot away any remaining liquid. a) Prepare a 5% stacking gel and evenly drop it into the reserved gaps. Select the comb according to the required channels and insert it, avoiding air bubbles as much as possible; b) Mix the aortic sample protein to be tested with the loading buffer according to the instructions (4:1), and place the EP tube containing the protein solution into the pot and boil for about 15 minutes to denature the aortic protein; c) Take the electrophoresis tank, correctly place the sandwich glass plate in the electrophoresis tank, add the freshly prepared electrophoresis solution, remove the comb, add 20 μl of the sample to be tested into the channels, and add 5 μl of the marker into the two side channels. Be careful to avoid air bubbles during the perfusion process; d) Connect the power supply, first select a constant voltage of 80V for electrophoresis for about 30 minutes until the band exceeds the gel boundary mark, then select a constant voltage of 120V until the blue band reaches the edge of the gel.

[0087] Transfer and blocking: a) Remove the sandwich glass plate from the electrophoresis tank and place it in a large beaker containing buffer solution. a) Cut a PVDF membrane to the appropriate size and activate it in a petri dish containing methanol. Remove it after 15 seconds. b) Soak the sponge and filter paper in a tray containing transfer buffer. Carefully remove the colloid and, based on the relative molecular weight of the target protein, refer to the marker label to determine the approximate location of the target protein and cut it. c) Spread the colloid evenly on the filter paper, then cover it with the labeled PVDF membrane. Remove any small air bubbles under the membrane, and cover it with filter paper and sponge in sequence to complete the "sandwich" construction. Place it correctly in the electroporation tank, place appropriately sized ice packs on both sides of the tank, add the buffer solution, and finally place the tank in a basin containing crushed ice. d) Connect to a 220V power supply and select a constant current of 250mA based on the relative molecular mass of the target protein. The process takes 1-1.5 hours. e) Dilute skim milk powder or bovine serum albumin with TBST to obtain a 5% blocking solution (bovine serum albumin is used as the blocking solution and antibody dilution solution for detecting phosphorylated proteins). Place the PVDF membrane in an incubation box containing the sealing solution, and place the incubation box on a horizontal shaker. The sealing process will be completed in about 1 hour (set the horizontal shaker to medium speed).

[0088] Antibody incubation and color development: a) Dilute the antibody with bovine serum albumin (5% BSA) according to the characteristics of different antibodies and experimental requirements. The dilution ratio needs to be adjusted gradually according to the experimental results. It is best to use the recommended antibody concentration at the beginning of the experiment. a) After sealing, place the PVDF membrane flat in a plastic bag, add the antibody, seal the protein band, and place it in the refrigerator's crisper drawer overnight at 4°C; b) Rinse the target protein bands overnight for 10-15 minutes each time, for a total of 3 rinses; c) After rinsing, add diluted secondary antibody to each target protein band and incubate on a horizontal shaker at room temperature for 1 hour; d) Return the secondary antibody to a centrifuge tube and rinse the protein bands 3 times, following the same procedure; e) After protecting the centrifuge tubes from light, add equal volumes of solutions A and B to the centrifuge tubes and mix to prepare the chemiluminescent solution; f) Turn on the UVP gel imaging system, place the bands in a dark room, add the chemiluminescent working solution evenly, and expose to obtain WB bands. Analyze the relative expression level of the target protein.

[0089] 2.5 Colon: (1) HE staining method to observe the pathological changes of colon tissue structure: Fresh colon tissue was fixed with 4% paraformaldehyde solution, paraffin tissue blocks were sliced, baked for 30 min (60℃), and dewaxed in xylene solution. Then the slices were placed in alcohol and distilled water with decreasing concentrations. After washing with alcohol and distilled water with decreasing concentrations, the slices were soaked in hematoxylin solution and then washed with distilled water to remove residual staining solution. The slices were separated by 1% hydrochloric acid alcohol for 30 s, and then washed in distilled water for about 5 min. The stained slices were placed in alcohol and xylene solutions with increasing concentrations. After removal, resin was dripped and the slices were mounted. After the slices dried, the pathological manifestations of colon tissue were observed under an electron microscope and photographed. (2) Immunohistochemistry of paraffin sections: multiple immunofluorescence staining of colon tissue occludin (blocking protein) and ZO-1 (tight junction protein), and the number of Alcian blue / periodic acid–Schiff (AB-PAS, mucin) goblet cells were recorded.

[0090] 2.6 Bone marrow infiltration: (1) Bone marrow sampling and smear & staining (to examine bone marrow infiltration): The mouse bone marrow was flushed out of the bone marrow cavity with PBS and placed on a glass slide. After the slide dried, a few drops of Wright's stain were added. After staining for 2 minutes, buffer solution was added at a ratio of Wright's stain: buffer solution 1:1.5. Air was blown onto the glass slide using an air bag to mix it thoroughly. After 15 minutes, the slide was gently rinsed with tap water and allowed to air dry naturally before being observed under a microscope. Bone marrow samples were fixed in formalin, embedded in paraffin, sectioned, dewaxed, and immunohistochemically stained.

[0091] 2.7 Tumor tissue and extramedullary infiltration (liver, spleen, lungs)

[0092] (1) Sampling and measurement of tumor tissue and organs: After euthanizing the mice, the long and short diameters of the tumor tissue, spleen, liver and kidneys were measured with vernier calipers, and their weights were recorded; V (mm) ３ = [long diameter L × short diameter W × short diameter W] / 2; The formula for calculating the volume of an irregular tumor is: V = π / 6 × L (long diameter) × W2 (short diameter) or V = π / 6 × L (long diameter) × W (short diameter) × H (height);

[0093] Tumor inhibition rate (%) = (average tumor weight in control group - average tumor weight in experimental group) / average tumor weight in control group × 100; a tumor growth inhibition rate <40% is considered ineffective; a tumor growth inhibition rate ≥40% and statistically analyzed with P<0.05 is considered effective;

[0094] Spleen index = mouse spleen weight (mg) / mouse body weight (g); Liver index = liver weight (100 g) / mouse body weight.

[0095] (2) HE staining to observe tumor cell infiltration and proliferation: Fresh tissue was fixed with 4% paraformaldehyde solution. Paraffin tissue blocks were sectioned, baked for 30 min (60℃), and dewaxed in xylene solution. The sections were then placed in alcohol and distilled water in a gradient of decreasing concentration. After washing with alcohol and distilled water in a gradient of decreasing concentration, the sections were immersed in hematoxylin solution and then washed with distilled water to remove residual staining solution. The sections were separated by 1% hydrochloric acid alcohol for 30 s, and then washed with distilled water for about 5 min. The stained sections were then placed in alcohol and xylene solutions in a gradient of increasing concentration. After removal, resin was applied and the sections were mounted. After the sections dried, the pathological features of the tissue were observed under an electron microscope and photographed.

[0096] To screen key strains with therapeutic potential in the gut of healthy individuals, this study used culture omics technology to isolate *Lactobacillus reuteri* HG001 from feces of healthy individuals. After assessing its probiotic properties, the in vivo antitumor activity of this bacterium was further evaluated. Results are as follows: Figure 1As shown, by administering this bacterium via gavage to mice bearing diffuse large B-cell lymphoma in combination with chemotherapy, we found that live *Lactobacillus reuteri* intervention did not affect the overall growth of the mice, indicating good safety. Compared with cyclophosphamide chemotherapy alone (MC group), the combination with *Lactobacillus reuteri* (MCL group) significantly inhibited tumor growth, as evidenced by reduced tumor volume (p = 0.0225) and weight (p = 0.0357), delayed tumor growth, and decreased levels of serum markers related to tumor burden, such as LDH, SF, and β2-MG. Furthermore, after the intervention, we collected feces from each group of mice for 16S rRNA sequencing. The results showed that the α- and β-diversity of the gut microbiota in the MCL group were higher than those in the MC group, and the abundance of *Lactobacillus* and Firmicutes increased. *Lactobacillus* abundance was negatively correlated with inflammation and tumor burden. These results indicate that *Lactobacillus reuteri* helps enhance the antitumor efficacy of cyclophosphamide against DLBCL.

[0097] Example 3: In vitro cell experiments to verify the effective components of the bacteria.

[0098] High-throughput sequencing and metabolomics sequencing of Lactobacillus reuteri HG001 were used to identify whether the effective metabolite is indole-3-carboxaldehyde (I3A) and its related mechanisms of action.

[0099] I. Cellular Experiments:

[0100] 1. Experimental Protocol: *Lactobacillus reuteri* in the logarithmic growth phase and *Lactobacillus reuteri* heat-inactivated at 100°C for 15 minutes were counted. Then, a hemocytometer was used to count the cells. Cells were plated at ratios of 1:1000, 1:100, 1:10, and 1:1 (SU-DHL-4, OCI-LY3) to bacteria. After 4 hours of intervention with appropriate bacterial concentrations, the cell proliferation inhibition rate was compared using a CCK8 assay. Similarly, bacterial cultures in the logarithmic growth phase were centrifuged and filtered to obtain the supernatant. This supernatant was then mixed with cell culture medium (PRMI-1640 complete medium) at different proportions (10%-60%). The mixed bacterial supernatant and medium were added to the cells for 4 hours of intervention. After intervention, the cell proliferation inhibition was compared using a CCK8 assay.

[0101] The SU-DHL-4 cell line was purchased from the Chinese Academy of Sciences Cell Bank, and the OCI-LY3 cells were purchased from Cybio (Shanghai) Biotechnology Co., Ltd.

[0102] II. Cell Experiments (I):

[0103] Objective: To demonstrate that the supernatant (metabolites) of bacteria can inhibit the proliferation of tumor cells in vitro.

[0104] 1. Experimental groups (total incubation for 3-4 hours):

[0105] Group C: DLBCL cells + cyclophosphamide + PBS;

[0106] CL group: DLBCL cells + cyclophosphamide + live Lactobacillus reuteri (Lactobacillus reuteri and cells were added at a 1:1 ratio);

[0107] CS group: DLBCL cells + cyclophosphamide + Lactobacillus reuteri supernatant (obtained by centrifugation at 8000 rpm for 5 min, aspirating the supernatant and filtering it through a 0.22 μm filter, then mixing it with cell culture medium at a ratio of 8:2);

[0108] CH group: DLBCL cells + cyclophosphamide + inactivated Lactobacillus reuteri (inactivated at 100℃ for 15 min);

[0109] 2. Detection indicators: (1) Comparison of cell proliferation capacity in each group: CCK8 assay. (2) Comparison of cell cycle changes and apoptosis in each group: flow cytometry.

[0110] III. Cell Experiments (II)

[0111] Objective: To verify whether the active ingredient in the supernatant is indole-3-carboxaldehyde (ICAld).

[0112] 1. Experimental groups (total incubation for 3-4 hours):

[0113] Con group: DLBCL cells + PBS;

[0114] Group I: DLBCL cells + ICA1d (0.5mM);

[0115] Group L: DLBCL cells + ILA (0.5mM);

[0116] Group A: DLBCL cells + XA (0.5mM);

[0117] ILA stands for indole-3-lactic acid, and XA stands for xanthuric acid.

[0118] 2. Detection indicators: (1) Comparison of cell proliferation capacity in each group: CCK8 assay; (2) Comparison of cell cycle changes and apoptosis in each group: flow cytometry; (3) Western blot detection of mechanism-related proteins: Cyclophosphamide pathway: Bax, Bcl-2, Caspase3, Cleaved-Caspase3. Metabolite-related pathways: AhR, AHR, p-PI3K, p-AKT, PTEN.

[0119] After confirming that *Lactobacillus reuteri* possesses an anti-DLBCL effect, we continued to investigate the main active components responsible for the synergistic anti-cancer effect of this strain. The study first explored the inhibitory effects of different components of *Lactobacillus reuteri* on two DLBCL cell lines (SU-DHL-4 and OCI-LY3). For example... Figure 2 As shown, live *Lactobacillus reuteri*, heat-inactivated bacterial cells, and bacterial supernatant were diluted to different proportions and co-incubated with tumor cells (4 h). The inhibitory efficiency against tumor cells was detected by the CCK8 assay. The results showed that the inhibitory efficiency of inactivated bacterial cells against DLBCL cells was concentration-independent and relatively poor; conversely, live bacteria and supernatant showed similar inhibitory efficiencies, exhibiting a concentration-dependent effect. Subsequently, cells treated with chemotherapy drug CTX were further co-cultured with different bacterial components, and the expression levels of apoptosis proteins were detected. The results also showed a similar pro-apoptotic trend in the bacterial supernatant and live bacteria. Based on these results, we believe that the main effective component of *Lactobacillus reuteri* against DLBCL is located in the bacterial supernatant, rather than the bacterial cells themselves. Simultaneously, we used Western blotting to detect apoptosis-related proteins in the treated tumor cells, finding that the expression of pro-apoptotic proteins was upregulated and the expression of anti-apoptotic proteins was decreased after intervention.

[0120] To identify key anti-DLBCL metabolites in the supernatant of *Lactobacillus reuteri*, we used targeted tryptophan metabolomics to detect these metabolites and analyzed their correlation with metabolites in the feces of DLBCL-bearing mice after *Lactobacillus reuteri* intervention. Figure 3 As shown, the results revealed that after gavage administration of *Lactobacillus reuteri*, the abundance of indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid in mouse feces significantly increased. The same metabolites were also detected in the bacterial supernatant, indicating that *Lactobacillus reuteri* is indeed the main source of these metabolites. Next, we treated the DLBCL cell line with different concentrations of indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid and observed their inhibitory effects on cell proliferation. The results showed that while the inhibition of DLBCL cells by the three metabolites all exhibited a time- and concentration-dependent trend, indole-3-carboxaldehyde had the most significant inhibitory effect, while xanthuric acid had the weakest inhibitory effect. Subsequently, the study continued by adding the three metabolites to the chemotherapy drug CTX for co-incubation to explore their sensitizing effect on chemotherapy. The results showed that regardless of whether chemotherapy was combined with indole-3-carboxaldehyde, it exhibited the most significant tumor-suppressive ability. The above results indicate that indole-3-carboxaldehyde is a key active metabolite of *Lactobacillus reuteri* that exerts its antitumor effects.

[0121] Example 4: Animal experiments to verify signaling pathways

[0122] Verify the regulatory mechanism by which the metabolite of *Lactobacillus reuteri* HG001 (indole-3-carboxaldehyde: I3A) improves the prognosis of DLBCL.

[0123] I. Animal Experiments (I)

[0124] 1. Experimental Groups:

[0125] MCL_neg group: Model + Cyclophosphamide + Lactobacillus reuteri + Tryptophan-deficient diet (DLBCL + CTX + bacteria).

[0126] MC group: model + cyclophosphamide + normal diet (DLBCL + CTX);

[0127] MCL group: model + cyclophosphamide + Lactobacillus reuteri + normal diet (DLBCL + CTX + Lactobacillus reuteri).

[0128] 2. Detection indicators: (1) Mouse-related indicators: mouse hair, luster, feeding status, mental state, weight change, subcutaneous tumor changes (photographs), survival curve, tumor growth curve. (2) Tumor-related indicators: Macroscopic indicators: tumor volume and weight, HE staining of tumor tissue; Tumor-related indicators: Ki67 (related to tumor cell proliferation), EZH2 (promotes tumor proliferation and differentiation), Bcl-2 (involved in apoptosis control). (3) Intestinal-related indicators: Macroscopic indicators: colon length, intestinal mucosal permeability; Pathological changes: (HE staining) Observe the pathological changes in mouse ileum and colon tissue (intestinal villi, inflammatory cells, glandular tissue, intestinal stem cells, intestinal mucosal lamina propria lymphocytes, etc.); Permeability-related: (WB) MUC2, ZO-1, ZO-2, claudin-1, occludin; Inflammation-related: (qPCR) NF-κB (pro-inflammatory), IL-1, IL-12 (pro-inflammatory), IL-6, TNF-α, IL-10 (anti-inflammatory), IL-22 (involved in mucosal repair).

[0129] (4) Other indicators: ELISA was used to detect the I3A content in feces, blood and tumor tissue; qPCR was used to detect the abundance changes of Lactobacillus in mouse feces; Western blot was used to detect cyclophosphamide pathway proteins: Bax, Bcl-2, CytC, Caspase3, Cleaved-Caspase3; Western blot was used to detect metabolite pathway proteins: AhR, Cydlal (AhR target gene).

[0130] To verify in vivo whether the tryptophan pathway is a key pathway for the production of indole-3-carboxaldehyde, we continued to feed DLBCL tumor-bearing mice with tryptophan-deficient diets (MCL_neg group) and standard diets (MCL group), respectively, while maintaining other treatments. Figure 4 As shown, the results indicated that compared to the MCL group, the MCL_neg group had increased tumor burden and inflammation levels, with a 5.37% increase in the proportion of Ki67 and P53 positive cells (p = 0.0011). Metabolomics analysis revealed decreased ICA1d levels in the MCL_neg group mice, and these levels were positively correlated with tryptophan intake. Regarding intestinal function, we also observed a shortened colon length in the MCL_neg group (p = 0.003). These findings confirm that indole-3-carboxaldehyde, produced by *Lactobacillus reuteri* through tryptophan metabolism, can inhibit DLBCL, and that tryptophan deficiency can reverse this effect and exacerbate barrier damage.

[0131] II. Animal Experiments (Part Two)

[0132] Experimental Groups:

[0133] MC group: Model + cyclophosphamide;

[0134] MCI_50 group: model + cyclophosphamide + ICA1d (50mM);

[0135] MCI_100 group: model + cyclophosphamide + ICA1d (100mM);

[0136] MCI_200 group: model + cyclophosphamide + ICA1d (200mM);

[0137] Detection indicators: (1) Mouse-related indicators: Same as animal experiment (I). (2) Tumor-related indicators: Macroscopic indicators: tumor volume and weight, tumor tissue HE staining; Tumor-related indicators: Ki67 (related to tumor cell proliferation), Bcl-2 (involved in apoptosis control). (3) Intestinal-related indicators: Macroscopic indicators: Same as animal experiment (I); Permeability-related indicators: (WB) MUC2, ZO-1, ZO-2, claudin-1, occludin; Inflammation-related indicators: (qPCR) NF-κB (pro-inflammatory), IL-1, IL-12 (pro-inflammatory), IL-6, TNF-α, IL-10 (anti-inflammatory), IL-22 (involved in mucosal repair). (4) Other indicators: Same as animal experiment (I).

[0138] To further evaluate the in vivo antitumor potential of indole-3-carboxaldehyde, we administered three different concentration gradients of indole-3-carboxaldehyde (50 mM, 100 mM, and 200 mM) to the drinking water of DLBCL tumor-bearing mice, with all other treatments remaining the same. After the intervention, we found that compared with the control group (MC group), indole-3-carboxaldehyde combination therapy significantly reduced tumor burden, manifested as a decrease in tumor volume and weight, a slowdown in tumor growth rate, and a reduction in the proportion of P53-positive cells. We then measured the concentration of indole-3-carboxaldehyde in mouse serum and analyzed its correlation with tumor burden. The results showed that the antitumor effect of indole-3-carboxaldehyde was significantly concentration-dependent, with the highest concentration group (MCI_200) showing the strongest effect. Serum indole-3-carboxaldehyde levels were positively correlated with the administered concentration and negatively correlated with tumor burden. In addition, indole-3-formaldehyde intervention can also help improve the colonic barrier structure and inflammatory status. The MCI_200 group had the largest colon length, the most goblet cells, the mildest inflammation, and the best barrier integrity, suggesting that indole-3-formaldehyde can alleviate chemotherapy-induced intestinal microenvironment disorder and barrier damage. Related analyses also support this conclusion.

[0139] III. Animal Experiments (Part Three)

[0140] Experimental Groups:

[0141] MC group: model + cyclophosphamide (30 mg / kg) + PBS;

[0142] MCI group: model + cyclophosphamide (30 mg / kg) + ICA1d (100 mM);

[0143] MCII group: Model + cyclophosphamide (30 mg / kg) + ICA1d (100 mM) + CH-223191 (5 mg / kg);

[0144] MCL group: model + cyclophosphamide (30 mg / kg) + Lactobacillus reuteri (100 μL / time, 10^9 CFU / mL, once every other day);

[0145] MCLI group: model + cyclophosphamide (30 mg / kg) + Lactobacillus reuteri (100 μL / dose, 10^9 CFU / mL, every other day) + CH-223191 (5 mg / kg);

[0146] Detection indicators: (1) Mouse-related indicators: same as animal experiment (I). (2) Tumor-related indicators: macroscopic indicators: same as animal experiment (I). (3) Intestinal-related indicators: same as animal experiment (I). (4) Other indicators: same as animal experiment (I).

[0147] To verify whether indole-3-carboxaldehyde, a major beneficial metabolite of *Lactobacillus reuteri*, also inhibits tumor proliferation by activating aryl hydrocarbon receptors to regulate related pathways, we conducted in vivo validation. We intervened in DLBCL-bearing mice by gavage with *Lactobacillus reuteri* or by adding indole-3-carboxaldehyde to drinking water, and included aryl hydrocarbon receptor antagonist groups. Results showed that compared with the chemotherapy group (MC), both indole-3-carboxaldehyde combined with chemotherapy (MCI group) and *Lactobacillus reuteri* combined with chemotherapy (MCL group) reduced tumor burden, manifested as tumor volume reduction, weight reduction, slowed growth, and decreased serum levels of LDH, SF, and β2-MG. The phenotype regulated by *Lactobacillus reuteri* and indole-3-carboxaldehyde is highly consistent, suggesting that indole-3-carboxaldehyde is a key active metabolite. Aromatic hydrocarbon receptor antagonist intervention (MCII and MCLI groups) increased the proportion of Ki67 and P53 positive cells in tumor tissue.

[0148] Further analysis showed that systemic inflammation was reduced in the MCI and MCL groups, while the AHR antagonist reversed this effect in the MCII and MCLI groups. Correlation analysis showed that AHR expression levels in tumor tissue were positively correlated with chemotherapy efficacy and negatively correlated with inflammatory burden. These in vivo results indicate that *Lactobacillus reuteri* and its metabolite indole-3-carboxaldehyde enhance the chemosensitivity of DLBCL to cyclophosphamide by activating aryl hydrocarbon receptors and improve chemotherapy-induced intestinal barrier dysfunction and inflammatory response.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri ), characterized in that, The Lactobacillus reuteri mentioned is Lactobacillus reuteri HG001, which was deposited on October 20, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.36248.

2. The use of *Lactobacillus reuteri* and / or its metabolites as described in claim 1 in the preparation of a medicament for treating diffuse large B-cell lymphoma, characterized in that... The metabolites of *Lactobacillus reuteri* are at least one of indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid.

3. The application according to claim 2, characterized in that, The applications include the development of drugs to improve the side effects of cyclophosphamide in the treatment of diffuse large B-cell lymphoma.

4. The application according to claim 3, characterized in that, The drug reduces gastrointestinal side effects of cyclophosphamide treatment for diffuse large B-cell lymphoma by at least one of the following methods: (1) Enhance the α- and β-diversity of gut microbiota; (2) Increase the abundance of Lactobacillus and Firmicutes in the gut; (3) Relieves intestinal microenvironment disorder and barrier damage caused by chemotherapy.

5. The application according to claim 2, characterized in that, The indole-3-carboxaldehyde is produced by *Lactobacillus reuteri* through tryptophan metabolism.

6. The use of *Lactobacillus reuteri* and / or its metabolites synergistically with cyclophosphamide as described in claim 1 in the preparation of a pharmaceutical combination for treating diffuse large B-cell lymphoma, characterized in that... The metabolites of *Lactobacillus reuteri* are at least one of indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid.

7. The application according to claim 6, characterized in that, The drug combination inhibits tumor growth through at least one of the following mechanisms: (1) The tumor volume and weight decreased; (2) Slowed tumor growth; (3) Decreased levels of serum markers related to tumor burden, including LDH, SF, and β2-MG.

8. The application according to claim 6, characterized in that, The indole-3-carboxaldehyde is produced by *Lactobacillus reuteri* through tryptophan metabolism. The *Lactobacillus reuteri* and its metabolite indole-3-carboxaldehyde can enhance the chemosensitivity of cyclophosphamide to diffuse large B-cell lymphoma by activating aryl hydrocarbon receptors.

9. A drug for treating diffuse large B-cell lymphoma, characterized in that, The drug comprises *Lactobacillus reuteri* as described in claim 1 and / or its metabolites, wherein the metabolites of *Lactobacillus reuteri* are at least one of indole-3-carboxaldehyde, indole-3-lactic acid, and xanthuric acid.

10. The medicament according to claim 9, characterized in that, The drug also includes cyclophosphamide.

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