Use of lactobacillus mucosae or equol in the preparation of a drug for the immunotherapy of sensitized colorectal cancer

Abstract

This invention discloses the application of *Lactobacillus mucosa* or equol in the preparation of sensitizing immunotherapeutic drugs for colorectal cancer. *Lactobacillus mucosa* LMSJ001 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29885. This invention utilizes *Lactobacillus mucosa* LMSJ001 to sensitize PD-1 antibodies against microsatellite stable (immunotherapy-insensitive) colorectal cancer mice. Prophylactic or therapeutic gavage significantly inhibits tumor growth in colorectal cancer mice. *Lactobacillus mucosa* LMSJ001 produces the metabolite equol, which sensitizes PD-1 antibodies against microsatellite stable (immunotherapy-insensitive) colorectal cancer mice, significantly inhibiting colorectal cancer both in vivo and in vitro, and promoting ferroptosis in colorectal cancer cells. Based on these unique functions, *Lactobacillus mucosa* LMSJ001 or its metabolite equol can be used synergistically or complementaryly with other methods to prepare pharmaceutical compositions, showing very broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically the application of *Lactobacillus mucosa* or its metabolite equol in the preparation of sensitized colorectal cancer immunotherapy products. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, with its incidence rising in both developed and developing countries. The pathogenesis of CRC is multifactorial, involving mutations in genes such as APC, KRAS, and TP53, environmental influences such as diet, smoking, and alcohol consumption, as well as chronic inflammation and gut microbiota dysbiosis. While advancements in endoscopic techniques, surgery, chemotherapy, and radiotherapy have been made in prevention and treatment, these methods carry significant side effects and high risks, reducing patients' quality of life. Therefore, there is an urgent need for novel treatment strategies that are both effective and have fewer side effects.

[0003] Ferroprelation is an iron-dependent form of cell death characterized by lipid peroxidation, reactive oxygen species accumulation, mitochondrial dysfunction, and iron metabolism disorders, ultimately leading to cell death. Studies have shown that inducing ferroptosis in tumor cells can effectively inhibit tumor development, particularly in drug-resistant and metastatic tumors, making it a novel strategy for cancer treatment. Research has found that antitumor drugs such as sorafenib can induce ferroptosis by promoting TRIM54-mediated FSP1 ubiquitination and degradation through the ERK pathway. Furthermore, ferroptosis inducers can reduce immune escape mechanisms in the tumor microenvironment (TME) and enhance the efficacy of immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies).

[0004] In recent years, with the deepening of cancer research, people have developed a strong interest in the potential role of probiotics in cancer prevention and treatment. Probiotics have significant potential in regulating the gut microbiota, reducing inflammatory responses, and directly or indirectly inhibiting tumor growth. Unlike traditional therapies, probiotics can be administered orally, resulting in higher safety, generally good tolerability, and a lower risk of adverse reactions. Furthermore, probiotics, when combined with chemotherapy and other treatment regimens, have great potential for enhancing drug sensitization and improving prognosis. Using probiotics can help develop safer and more natural treatment options for colorectal cancer patients.

[0005] Limosilactobacillus mucosa (L. muc) is a relatively recently discovered probiotic, initially isolated from the intestinal mucosa of pigs and later found in the human gut. Limosilactobacillus mucosa can adhere tightly to the intestinal mucosal surface, possessing the potential to colonize the intestine and regulate intestinal barrier function. Studies have found that Limosilactobacillus mucosa can inhibit intestinal inflammatory responses and alleviate diarrhea, but its role in colorectal cancer remains unclear. Summary of the Invention

[0006] The first objective of this invention is to provide a novel strain of *Lactobacillus mucosa*.

[0007] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] A type of Lactobacillus mucosa, isolated from the feces of a healthy elderly person, named Lactobacillus mucosa LMSJ001, is deposited 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.29885 and deposit date of February 26, 2024.

[0009] *Limosilactobacillus mucosae* LMSJ001 was isolated from fecal samples of healthy elderly individuals in Lishui, Zhejiang Province, a region known for its longevity. 16S rDNA sequence analysis and nucleic acid sequence alignment using NCBI revealed that this strain exhibited the highest nucleic acid sequence similarity (nearly 100%) with *Limosilactobacillus mucosae*, suggesting that it belongs to the genus *Limosilactobacillus*. Whole-genome sequencing of this strain and comparison with the average nucleotide similarity of its genome with that of other *Limosilactobacillus* strains with published whole-genome sequences on NCBI showed significant differences between the genomes of this strain and those of previously published *Limosilactobacillus* strains, thus identifying it as a novel *Limosilactobacillus* strain.

[0010] The second objective of this invention is to provide the application of Lactobacillus mucosa LMSJ001 in the preparation of immunotherapeutic drugs for sensitized microsatellite-stabilized (immunotherapy-insensitive) colorectal cancer.

[0011] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0012] 1) Lactobacillus mucosa LMSJ001 significantly reduced the tumor size and weight of CT26 subcutaneous tumors;

[0013] 2) Lactobacillus mucosa LMSJ001 significantly enhances the efficacy of PD-1 antibody against CT26 subcutaneous tumors;

[0014] The third objective of this invention is to provide the application of Lactobacillus mucosa LMSJ001 in the preparation of drugs for the prevention and treatment of colorectal cancer.

[0015] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0016] 1) Prophylactic gavage with LMSJ001 significantly reduced the size and weight of MC38 subcutaneous tumors;

[0017] 2) Lactobacillus mucosa LMSJ001 significantly reduced the tumor size and weight of MC38 subcutaneous tumors;

[0018] 3) Lactobacillus mucosa LMSJ001 significantly increased CD8+ T cell infiltration in MC38 subcutaneous tumors.

[0019] The fourth objective of this invention is to provide the application of equol, a metabolite of Lactobacillus mucosa J001, in the preparation of sensitized microsatellite-stabilized (immunotherapy-insensitive) immunotherapeutic drugs for colorectal cancer.

[0020] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0021] 1) Lactobacillus mucosa LMSJ001 can be converted to produce the metabolite equadol;

[0022] 2) Estrol significantly reduced the size and weight of CT26 subcutaneous tumors;

[0023] 3) Estrogen significantly enhances the efficacy of PD-1 antibody in treating CT26 subcutaneous tumors;

[0024] The fifth objective of this invention is to provide the use of estrol, a metabolite of Lactobacillus mucosa J001, in the preparation of drugs for treating colorectal cancer.

[0025] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0026] 1) Estrol significantly reduced the size and weight of MC38 subcutaneous tumors;

[0027] 2) Estrol significantly inhibited the proliferation of human colon cancer cells HCT116 and HT29;

[0028] 3) Estrol significantly promotes apoptosis in HCT116 cells;

[0029] 4) Estrol significantly inhibited the cell cycle of HCT116 cells;

[0030] 5) Estrol can enter HCT116 cells to exert a tumor-suppressive effect.

[0031] The sixth objective of this invention is to provide the use of equol, a metabolite of Lactobacillus mucosa J001, in the preparation of a drug that promotes ferroptosis in colorectal cancer.

[0032] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0033] 1) Estrol significantly increased the reactive oxygen species level in HCT116 cells;

[0034] 2) Estrol significantly reduced the mitochondrial membrane potential level in HCT116 cells;

[0035] 3) Estrol significantly promotes ferroptosis in HCT116 cells, especially when combined with the ferroptosis inducer RSL3, it can significantly inhibit cell proliferation;

[0036] 4) Estrol-induced HCT116 cell death can be inhibited by ferroptosis inhibitors;

[0037] 5) Estrol significantly increases the level of lipid peroxides in HCT116 cells, and this effect can be inhibited by the ferroptosis inhibitor Fer-1.

[0038] The seventh objective of this invention is to provide a PD-1 inhibitor sensitizer, a formulation containing Lactobacillus mucosa LMSJ001 and / or equol, thereby sensitizing microsatellite stable colorectal cancer immunotherapy.

[0039] As a preferred embodiment of the present invention, the drug or sensitizer includes Lactobacillus mucosa LMSJ001 or its metabolite equadol, a drug carrier and / or pharmaceutically acceptable excipients.

[0040] As a preferred embodiment of the present invention, the dosage form of the drug or sensitizer is pills, tablets, powders, capsules, granules, suspensions, injections, oral liquids, enemas, or tube feeding preparations.

[0041] This invention provides the application of Lactobacillus mucosa LMSJ001 or its metabolite equol in the preparation of products related to the prevention and / or sensitization of colorectal cancer immunotherapy, which has at least the following advantages and beneficial effects:

[0042] 1) There are currently no relevant literature or patent reports on the immunotherapy of microsatellite-sensitized (immunotherapy-insensitive) colorectal cancer by LMSJ001 mucosa or its metabolite equol, which provides a new method for enhancing the efficacy of tumor immunotherapy.

[0043] 2) Lactobacillus mucosa LMSJ001 has a significant preventive and therapeutic effect on the occurrence and development of colorectal cancer and can be used as a beneficial supplement to probiotic therapy.

[0044] 3) Estrol significantly inhibits colorectal cancer, promotes tumor cell apoptosis, inhibits the cell cycle, and promotes tumor cell ferroptosis, providing new clues and ideas for future research on methods to inhibit tumors. Attached Figure Description

[0045] Figure 1The image shows the whole genome sequencing diagram of Lactobacillus mucosa LMSJ001 and a comparison diagram with the whole genomes of known Lactobacillus mucosa strains whose whole genome sequences have been published by NCBI. In the figure: A is the genome circle diagram of Lactobacillus mucosa LMSJ001; B is the average nucleotide similarity analysis diagram between Lactobacillus mucosa LMSJ001 and known Lactobacillus mucosa strains whose whole genome sequences have been published by NCBI.

[0046] Figure 2 This image illustrates the efficacy of immunotherapy in microsatellite-sensitized (immunotherapy-insensitive) colorectal cancer patients treated with LMSJ001. In the image: A shows the tumor size in CT26 subcutaneous tumor mice treated with LMSJ001 and PD-1 antibodies alone and in combination; B shows the tumor growth curves in CT26 subcutaneous tumor mice treated with LMSJ001 and PD-1 antibodies alone and in combination.

[0047] Figure 3 The diagram illustrates the significant inhibitory effect of prophylactic gavage of Lactobacillus mucosa LMSJ001 on tumor growth in a mouse model of MC38 subcutaneous tumor. In the diagram: A is a schematic diagram of modeling; B is a tumor growth curve; C is a schematic diagram of tumor size; D is a statistical graph of tumor weight; and E is a statistical graph of the abundance of Lactobacillus mucosa LMSJ001 in mouse feces.

[0048] Figure 4 The figure illustrates the significant inhibitory effect of LMSJ001 on tumor growth in a mouse model of MC38 subcutaneous tumor. In the figure: A is a schematic diagram of model establishment; B is a tumor growth curve; C is a schematic diagram of tumor size; D is a statistical graph of tumor weight; E is a graph showing tumor infiltration into CD8+. + T cell proportion statistics chart.

[0049] Figure 5 Metabolomics analysis and mass spectrometry detection of equol, a metabolite produced by *Lactobacillus mucosa* LMSJ001, are shown in the figure. Figure A shows a volcano plot of differential metabolites in mouse serum after gavage of *Lactobacillus mucosa* LMSJ001 compared to the control group; Figure B shows a heatmap (left) and VIP value plot (right) of differential metabolite expression in mouse serum after gavage of *Lactobacillus mucosa* LMSJ001 compared to the control group; Figure C shows the equol content in the feces of mice in the *Lactobacillus mucosa* LMSJ001 gavage group and the control group; Figure D shows the equol content in the culture supernatant and blank culture medium of *Lactobacillus mucosa* LMSJ001 cultured in vitro using M9 medium supplemented with daidzein (DZN).

[0050] Figure 6This image illustrates the effect of equol-sensitized microsatellite stable (immunotherapy-insensitive) colorectal cancer immunotherapy. In the image: A is a schematic diagram of tumor size in CT26 subcutaneous tumor mice treated with equol and PD-1 antibody alone and in combination; B is a tumor growth curve in CT26 subcutaneous tumor mice treated with equol and PD-1 antibody alone and in combination.

[0051] Figure 7 The diagram illustrates the significant inhibitory effect of equol on colon cancer. In the diagram, A shows the tumor size of mice with significantly inhibited MC38 subcutaneous tumors after equol intervention; B shows the tumor growth curve of mice with significantly inhibited MC38 subcutaneous tumors after equol intervention; C shows the tumor weight of mice with significantly inhibited MC38 subcutaneous tumors after equol intervention; D shows the proliferation of HCT116 and HT29 cells after intervention with different concentrations of equol using the CCK8 assay; E shows the colony formation of HCT116 and HT29 cells after equol intervention using a plate colony assay; F shows the colony formation count of HCT116 and HT29 cells after equol intervention using a plate colony assay; G shows the apoptosis rate of HCT116 cells after equol intervention; H shows the cell cycle of HCT116 cells after equol intervention; and I shows the subcellular fluorescence localization of equol in HCT116 cells, where green represents the cell membrane, red represents CY5-labeled equol, and blue represents the cell nucleus.

[0052] Figure 8 The diagram illustrates the effect of equol on ferroptosis in colorectal cancer. In the diagram: A shows the reactive oxygen species (ROS) level in HCT116 cells after equol intervention; B shows the mitochondrial membrane potential level in HCT116 cells after equol intervention; C shows the doubling of HCT116 cells after equol intervention under the action of the ferroptosis inducer RSL3; D shows the doubling of HCT116 cells after equol intervention under the action of the ferroptosis inhibitor Fer-1; E shows the lipid peroxide levels in HCT116 cells after intervention with equol, equol, and the ferroptosis inhibitor Fer-1. Detailed Implementation

[0053] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0054] Example 1: Isolation, screening and identification of Lactobacillus mucosa LMSJ001

[0055] (1) Strain isolation: In an anaerobic workstation, 2g of fecal samples from healthy elderly people in Lishui, Zhejiang, known as the hometown of longevity, were taken, serially diluted with sterile physiological saline, and spread onto RCM agar plates and anaerobically cultured at 37℃. Single colonies were selected for purification culture.

[0056] (2) Enrichment of strains: The purified strains were inoculated one by one into sterile RCM broth medium on a sterile operating table using an inoculation loop, placed in a shaker, and cultured at 37°C for 48 hours before 16S rDNA sequencing identification.

[0057] (3) 16S rDNA sequencing identification: Colony PCR amplification of strain LMSJ001 was performed using universal bacterial primers and sent to Hangzhou Mingke Biotechnology Co., Ltd. for 16S rDNA sequencing identification. The 16S rDNA of this strain was compared with the sequence on NCBI and found to be 1405bp in length. The sequence is as follows. It was found that the 16S rDNA sequence of Limosilactobacillus mucosae showed the highest homology (the similarity was about 100%), and the strain was identified as Limosilactobacillus mucosae.

[0058]

[0059]

[0060] (4) Whole genome identification: The LMSJ001 *Lactobacillus mucosa* was further sequenced at Shanghai Meiji Biotechnology Co., Ltd. The sequencing results were analyzed using Circos genome mapping, phylogenetic tree analysis, and pan-genome Venn diagram analysis. The results are as follows: Figure 2 A is the completed genome sequencing diagram of strain LMSJ001 (Circos genome diagram analysis); as... Figure 2 As shown in B, the average nucleotide similarity (ANI) value is calculated by comparing homologous regions between genomes, reflecting genomic similarity. Generally, genomes with an ANI value higher than 99.9% are considered to be from the same strain. *Lactobacillus mucosa* LMSJ001 differs from other known reported *Lactobacillus mucosa* strains. Based on the above results and a literature review, this *Lactobacillus mucosa* strain LMSJ001 is different from previously reported strains and is a newly discovered *Lactobacillus mucosa* strain.

[0061] This strain was deposited on February 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC NO.29885; suggested taxonomic name: Limosilactobacillus mucosae.

[0062] Example 2: Immunotherapy for colorectal cancer sensitized by LMSJ001-sensitized microsatellite stable (immunotherapy-insensitive) Lactobacillus mucosa

[0063] Six-week-old male Balb / c mice were randomly divided into four groups of 10 mice each. Mice were given free access to drinking water containing 0.2 mg / mL ampicillin, neomycin sulfate, metronidazole, and 0.1 mg / mL vancomycin for three days to initially clear the intestinal flora. Mice were then subcutaneously injected with a mixture of CT26 cells and matrix gel (cell to matrix gel ratio 1:1) at a dose of 10. 6 Cells / mouse, injection volume 100 μL / mouse. Four groups of mice were treated with PBS, *Lactobacillus mucosa* LMSJ001, PBS + PD-1 antibody, and *Lactobacillus mucosa* LMSJ001 + PD-1 antibody, respectively. PBS and *Lactobacillus mucosa* LMSJ001 were administered by gavage at a dose of 1 × 10⁻⁶. 9 CFU / mouse, 200 μL / mouse, once daily via gavage; PD-1 antibody, 100 μg / mouse, administered intraperitoneally on days 7, 10, and 13. Tumor size was measured every two days during modeling, and tumor size and weight were recorded after modeling was completed.

[0064] The results are as follows Figure 2 As shown, PD-1 antibody treatment alone has little effect, while Lactobacillus mucosa LMSJ001 treatment alone, especially in combination with PD-1 antibody, can significantly reduce tumor size in CT26 subcutaneous tumor mice.

[0065] Example 3: Prophylactic gavage of Lactobacillus mucosa LMSJ001 significantly inhibited tumor growth in an MC38 subcutaneous tumor mouse model.

[0066] Six-week-old male C57BL / 6 mice were randomly divided into two groups of 10 mice each. The modeling process is as follows: Figure 3 As shown in Figure A, mice were allowed free access to drinking water containing 0.2 mg / mL ampicillin, neomycin sulfate, metronidazole, and 0.1 mg / mL vancomycin for 3 days to initially clear the intestinal flora, which was beneficial for colonization of transplanted bacteria. After intestinal flora clearance, both groups were administered PBS and Lactobacillus mucosa LMSJ001 by gavage at a dose of 1×10⁻⁶, respectively. 9 CFU / mouse, 200 μL / mouse, once daily via gavage. Five days after gavage, mice were subcutaneously injected with a mixture of MC38 cells and matrix gel (cell to matrix gel ratio 1:1), at a dose of 10... 6 Cells / mouse, injection volume 100 μL / mouse, this is recorded as day 0 (DAY0). Gavage was continued once daily until day 21. During the modeling process, tumor size was measured every 2 days. After modeling, tumor size and weight were recorded, and mouse feces were collected. The abundance changes of *Lactobacillus mucosa* LMSJ001 in the feces were detected using qPCR technology.

[0067] The results are as follows Figure 3As shown in Figure A, the modeling diagram demonstrates that *Lactobacillus mucosa* LMSJ001 significantly reduced tumor size (B and C) and tumor weight (D) in the MC38 subcutaneous tumor mouse model. Compared to the control group, the abundance of *Lactobacillus mucosa* LMSJ001 was significantly increased in the feces of mice in the *Lactobacillus mucosa* LMSJ001 gavage group (E).

[0068] Example 4: Lactobacillus mucosa LMSJ001 significantly inhibited tumor growth in an MC38 subcutaneous tumor mouse model.

[0069] Six-week-old male C57BL / 6 mice were randomly divided into two groups of 10 mice each. The modeling process is as follows: Figure 4 As shown in Figure A, mice were allowed free access to drinking water containing 0.2 mg / mL ampicillin, neomycin sulfate, metronidazole, and 0.1 mg / mL vancomycin for 3 days to initially clear the intestinal flora, which was beneficial for colonization of the transplanted bacteria. Mice were then subcutaneously injected with a mixture of MC38 cells and matrix gel (cell to matrix gel ratio of 1:1) at a dose of 10... 6 Cells / animal, injection volume 100 μL / animal, this is recorded as day 0 (DAY0). Both groups were administered PBS and Lactobacillus mucosa LMSJ001 by gavage, respectively, at a dose of 1 × 10⁻⁶. 9 CFU / mouse, 200 μL / mouse, once daily until day 21. During the modeling process, tumor size was measured every 2 days. After the modeling was completed, the size and weight of the tumor were recorded, and mouse tumor tissue was collected for flow cytometry to detect the proportion of CD8+ T cells in the tumor. In addition, mouse serum and feces were collected for further analysis.

[0070] The results are as follows Figure 4 As shown in Figure A, the modeling diagram shows that Lactobacillus mucosa LMSJ001 can significantly reduce the tumor size (B and C) and tumor weight (D) in MC38 subcutaneous tumor mice. Flow cytometry analysis shows that Lactobacillus mucosa LMSJ001 can significantly increase the proportion of infiltrating CD8+ T cells in the tumor (E).

[0071] Example 5: Metabolomics analysis and mass spectrometry detection of equol, a metabolite produced by Lactobacillus mucosa LMSJ001

[0072] Metabolomics analysis was performed on mouse serum collected in Example 3 to analyze differential metabolites in mouse serum after gavage administration of *Lactobacillus mucosa* LMSJ001. The content of equol in the feces of mice collected in Example 3 was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in the feces of mice in the *Lactobacillus mucosa* LMSJ001 gavage group and the control group. *Lactobacillus mucosa* LMSJ001 was cultured in vitro on M9 medium supplemented with the equol precursor daidzein (DZN), and the equol content in its culture supernatant and blank medium was detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0073] The results are as follows Figure 5 As shown, compared with the control group, the level of equol in mouse serum was significantly upregulated after gavage with LMSJ001 (A and B); compared with the control group, the level of equol in mouse feces was significantly increased after gavage with LMSJ001 (C); compared with the blank culture medium, the level of equol in the in vitro culture fermentation broth of LMSJ001 was significantly increased (D).

[0074] Example 6: Equadol-sensitized microsatellite-stabilized (immunotherapy-insensitive) colorectal cancer immunotherapy

[0075] Six-week-old male Balb / c mice were randomly divided into four groups of 10 mice each. Mice were subcutaneously injected with a mixture of CT26 cells and matrix gel (cell to matrix gel ratio of 1:1) at a dose of 10... 6 Cells / mouse, injection volume 100 μL / mouse. Four groups of mice were established: control group, equol group, control + PD-1 antibody group, and equol + PD-1 antibody group. The control solvent and equol were administered via gavage, with an intervention dose of 10 mg / kg mouse body weight and a gavage volume of 200 μL / mouse. Control mice were administered an equal volume of sodium carboxymethyl cellulose solution via gavage. The PD-1 antibody was administered via intraperitoneal injection at a dose of 100 μg / mouse, administered once each on days 7, 10, and 13. Tumor size was measured every two days during the modeling process.

[0076] The results are as follows Figure 6 As shown, estrol alone, especially in combination with PD-1 antibody, can significantly reduce tumor size in CT26 subcutaneous tumor mice.

[0077] Example 7: Equadol significantly inhibits colon cancer

[0078] In in vivo experiments, 6-week-old male C57BL / 6 mice were randomly divided into two groups of 10 mice each. Mice were subcutaneously injected with a mixture of MC38 cells and matrix gel (cell to matrix gel ratio of 1:1) at a dose of 10... 6Cells / mouse, injection volume 100 μL / mouse. Two groups were administered the control solvent and equol via gavage, respectively. The intervention dose of equol was 10 mg / kg mouse body weight, gavage volume 200 μL / mouse. The control group mice were administered an equal volume of sodium carboxymethyl cellulose solution via gavage once daily until day 21. During the modeling process, tumor size was measured every two days. After the modeling was completed, tumor size and weight were recorded.

[0079] In in vitro experiments, human colon cancer cells HCT116 and HT29 were resuscitated and cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum as a complete medium at 37°C and 5% CO2.

[0080] For cell proliferation assays, cells were seeded in 96-well plates at 1000 cells per well. After cell attachment, different concentrations of equol (0, 50 μM, 100 μM, 200 μM, and 300 μM) were added, with at least three replicates per group. Cells were cultured for 5 consecutive days. Each day, one plate was aspirated, and 100 μL of complete culture medium containing 10% CCK8 solution was added to each well. After incubation in the dark for 1.5 hours, the absorbance at 450 nm (OD450nm) was measured.

[0081] For plate cloning experiments, cells were seeded in 6-well plates with 1000 cells per well. After cell attachment, control solvent and 200 μM equaphen were added, with at least 3 replicates per group. After clonal plaques appeared, cells were fixed with 4% paraformaldehyde solution for 30 minutes, then stained with 0.1% crystal violet solution for 20 minutes. After washing with PBS, cells were air-dried in a clean bench, photographed with a digital camera, and the clonal plaques were counted and analyzed using ImageJ software, which can reflect the proliferation potential of individual cells.

[0082] For apoptosis detection, cells were seeded in 6-well plates, with 10 cells seeded per well. 5 Cells were cultured in batches, and after cell adhesion, control solvent and 200 μM equadol were added, with at least three replicates per group. After 48 hours of culture, cells were collected, and apoptosis was detected using the Annexin V-FITC / PI double staining method. The specific steps were as follows: cells were washed twice with pre-cooled PBS, resuspended in binding buffer, and then Annexin V-FITC and PI staining solutions were added. The cells were incubated in the dark for 15 minutes. Finally, the apoptosis rate was detected by flow cytometry, and the proportion of early apoptotic (Annexin V+ / PI-) and late apoptotic (Annexin V+ / PI+) cells was analyzed.

[0083] For cell cycle assays, cells were seeded in 6-well plates, with 10 cells seeded per well. 5Cells were cultured in batches, and after cell adhesion, control solvent and 200 μM equol were added, with at least three replicates per group. After 48 hours of culture, cells were collected, washed twice with pre-cooled PBS, and fixed with 70% ethanol overnight at 4°C. After fixation, cells were washed with PBS, and PI staining solution containing RNase A was added, followed by incubation in the dark for 30 minutes. Finally, flow cytometry was used to detect cell cycle distribution and analyze the proportions of cells in G0 / G1, S, and G2 / M phases to assess the effect of equol on cell cycle progression.

[0084] For subcellular fluorescence localization detection, cells were seeded in confocal microplates. After cell adhesion, CY5-labeled equadol was added and incubated at 37°C for 2 hours. The culture medium was discarded, and the cells were washed three times with PBS. Green fluorescently labeled cell membrane Dio probes were added and incubated at 37°C in the dark for 20 minutes. After washing with PBS, Hoechst 33342 staining solution was added and the cells were incubated at 37°C for 10 minutes to label the cell nuclei. The cells were then observed and images were acquired under a laser confocal microscope.

[0085] The results are as follows Figure 7 As shown, compared to the control group, equol intervention significantly reduced tumor size (A and B) and tumor weight (C) in MC38 subcutaneous tumor mice. Compared to the control group, equol intervention significantly inhibited the proliferation (D) and clonogenic ability (E and F) of HCT116 and HT29 colony cancer cells, significantly promoted apoptosis (G) of HCT116 cells, and inhibited the cell cycle of HCT116 cells (H). Subcellular fluorescence localization showed that equol could enter cells and localize in the cytoplasm (I).

[0086] Example 8: Estrol promotes ferroptosis in colorectal cancer

[0087] Human colon cancer cells HCT116 were resuscitated and cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum as a complete medium at 37°C in a 5% CO2 incubator. For the detection of reactive oxygen species (ROS) levels, cells were seeded into 6-well plates with 10 cells per well. 5 Cells were cultured in 6-well plates. After cell attachment, control solvent and 200 μM equadol were added, with at least 3 replicates per group. After 48 hours of culture, cells were collected and resuspended in serum-free medium containing 10 μM DCFH-DA probe, and incubated at 37°C in the dark for 30 minutes. Cells were washed three times with PBS, and the green fluorescence intensity (excitation / emission wavelength: 488 / 525 nm) was detected by flow cytometry to quantify ROS levels. Data were analyzed using FlowJo software. For mitochondrial membrane potential detection, cells were seeded in 6-well plates, with 10 cells per well. 5Cells were cultured in 96-well plates. After cell attachment, control solvent and 200 μM equol were added, with at least three replicates per group. After 48 hours of culture, cells were collected and JC-1 staining working solution (5 μg / mL) was added. The plates were incubated at 37°C in the dark for 20 minutes. After washing with PBS, the ratio of JC-1 monomers (green fluorescence, excitation / emission: 488 / 530 nm) to aggregates (red fluorescence, excitation / emission: 488 / 590 nm) was determined by flow cytometry, and the proportion of cells with decreased mitochondrial membrane potential was calculated. For cell doubling analysis using ferroptosis inducer (RSL3) combined with equol, cells were seeded at 1000 cells per well. After cell attachment, the cells were divided into three groups, with 0 μM, 0.5 μM, and 1 μM RSL3 added, respectively. Each group was further divided into two subgroups, with control solvent and 200 μM equol added, respectively. A control group was also included. Each group had at least three replicates. On days 0 and 4 of culture, the culture medium was aspirated, and 100 μL of complete culture medium containing 10% CCK8 solution was added to each well. After incubation in the dark for 1.5 hours, the absorbance at 450 nm (OD450nm) was measured. The cell doubling rate was calculated using the formula: cell doubling rate = log2(N / N0), where N is the current cell number and N0 is the initial cell number. For cell doubling rate analysis using the ferroptosis inhibitor (Fer-1) in combination with equadolphin, the grouping settings were the same as above, except that RSL3 was replaced with the ferroptosis inhibitor Fer-1 (0 μM, 0.5 μM, 1 μM), and the remaining treatments were the same as above. For lipid peroxide level detection, cells were seeded in 6-well plates, with 10 cells seeded per well. 5 Cells were collected and, after cell adhesion, were divided into three experimental groups: control group, equol group (200 μM), and equol + Fer-1 group (200 μM equol + 1 μM Fer-1). Each group had at least three replicates. After 48 hours of treatment, cells were collected, and C11-BODIPY 581 / 591 probe (2 μM) was added. Cells were incubated at 37°C in the dark for 30 minutes. After washing with PBS, flow cytometry was used to detect the ratio of the oxidized state (green fluorescence, excitation / emission: 488 / 510 nm) to the non-oxidized state (red fluorescence, excitation / emission: 488 / 590 nm) of the probe to quantify lipid peroxidation levels.

[0088] The results are as follows Figure 8As shown, compared to the control group, equol intervention significantly increased the reactive oxygen species level (ROS) in HCT116 cells (A) and significantly decreased the mitochondrial membrane potential level (B). The doubling rate of HCT116 cells after equol intervention was significantly reduced under the action of the ferroptosis inducer RSL3 (C). The doubling rate of HCT116 cells, which was reduced after equol intervention, was restored under the action of the ferroptosis inhibitor Fer-1 (D). Compared to the control group, the lipid peroxide level in HCT116 cells was significantly increased after equol intervention (E), and this effect could be inhibited by the ferroptosis inhibitor Fer-1 (E).

[0089] The above specific embodiments are used to explain and illustrate the present invention, and are only preferred embodiments of the present invention, not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. Mucosal Lactobacillus ( Limosilactobacillus mucosae LMSJ001, characterized in that, The Lactobacillus mucosa is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.29885.

2. The use of the *Lactobacillus mucosa* as described in claim 1 in the preparation of sensitized microsatellite-stabilized immunotherapy drugs for colorectal cancer.

3. The use of the *Lactobacillus mucosa* as described in claim 1 in the preparation of a drug for treating microsatellite-stabilized colorectal cancer.

4. A PD-1 inhibitor sensitizer, characterized in that, Containing the *Lactobacillus mucosa* as described in claim 1, or further containing its metabolite equol, thereby sensitizing microsatellite-stabilized colorectal cancer immunotherapy.

5. The application as described in any one of claims 2-3 or the sensitizer as described in claim 4, characterized in that, The dosage form of the drug or the sensitizer is pills, tablets, powders, capsules, granules, suspensions, injections, oral liquids, enemas, or tube feeding preparations.

6. The application as described in any one of claims 2-3 or the sensitizer as described in claim 4, characterized in that, The drug or the sensitizer includes a drug carrier and / or pharmaceutically acceptable excipients.

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