Application of lactobacillus mucosa or equol in preparation of sensitizing colorectal cancer immunotherapy medicine

Abstract

The invention discloses application of lactobacillus mucosa or equol in preparation of a sensitizing colorectal cancer immunotherapy drug. Lactobacillus mucosa LMSJ001 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.29885. The lactobacillus mucosa LMSJ001 is preserved in the China General Microbiological Culture Collection Center (CGMCC). According to the application disclosed by the invention, the curative effect of the PD-1 antibody on microsatellite stable (immunotherapy insensitive) colorectal cancer mice is enhanced through the lactobacillus mucosa LMSJ001, and the tumor growth of the colorectal cancer mice can be remarkably inhibited through prophylactic or therapeutic gavage. The lactobacillus mucosa LMSJ001 can generate a metabolite equol, the equol can sensitize the curative effect of a PD-1 antibody on microsatellite stable (immunotherapy insensitive) colorectal cancer mice, the colorectal cancer is remarkably inhibited in vivo and in vitro, and ferroptosis of colorectal cancer cells is promoted. Based on the unique functions, the lactobacillus mucosa LMSJ001 or a metabolite equol of the lactobacillus mucosa LMSJ001 can be cooperated or complemented with other means, can be used for preparing a pharmaceutical composition, and has a very wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to the application of mucosal lactobacillus or its metabolite equol in the preparation of a sensitized colorectal cancer immunotherapy product. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignancies worldwide, with an increasing incidence 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; and chronic inflammation and gut microbiome dysbiosis. Despite advances in prevention and treatment, endoscopic techniques, surgery, chemotherapy, and radiotherapy often present significant side effects and risks, impairing patients' quality of life. Therefore, new treatment strategies that are both effective and minimize side effects are urgently needed.

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

[0004] In recent years, with the deepening of cancer research, there has been a surge of interest in the potential role of probiotics in cancer prevention and treatment. Probiotics have significant potential to modulate the gut microbiome, reduce inflammation, and directly or indirectly inhibit tumor growth. Unlike traditional therapies, probiotics can be administered orally, offering improved safety, generally good tolerance, and a lower risk of adverse reactions. Furthermore, probiotics have great potential to enhance drug sensitivity and improve prognosis when combined with chemotherapy and other treatment options. The use of probiotics could potentially develop safer and more natural treatment options for patients with colorectal cancer.

[0005] Lactobacillus mucosae (L.muc) is a relatively recently discovered probiotic, originally isolated from the intestinal mucosa of pigs and later found in the human intestine. L.muc can tightly adhere to the intestinal mucosal surface and has the potential to colonize and modulate intestinal barrier function. Studies have shown that L.muc can inhibit intestinal inflammation and relieve diarrhea, but its role in colorectal cancer remains unclear. Summary of the Invention

[0006] The first object of the present invention is to provide a new strain of Lactobacillus mucosa.

[0007] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A mucosal lactobacillus was isolated from the feces of healthy elderly people and named Lactobacillus mucosalis LMSJ001. It was deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.29885 and the deposit date is February 26, 2024.

[0009] Lactobacillus mucosae (LMSJ001) was isolated from a stool sample of healthy elderly people in Lishui, Zhejiang Province, known as a "longevity village." 16S rDNA sequence analysis and nucleotide sequence alignment at NCBI revealed the highest nucleotide sequence similarity (nearly 100%) with L. mucosae, suggesting that it belongs to the genus Lactobacillus mucosae. Whole-genome sequencing of this strain and average nucleotide similarity analysis compared with all other L. mucosae strains with published full-genome sequences at NCBI revealed significant differences in the genomes of this strain from all previously published L. mucosae strains, identifying it as a novel L. mucosae strain.

[0010] The second object of the present invention is to provide the use of Lactobacillus mucosa LMSJ001 in the preparation of sensitized microsatellite stable (immunotherapy-insensitive) colorectal cancer immunotherapy drugs.

[0011] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

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

[0014] The third object of the present invention is to provide the use of Lactobacillus mucosa LMSJ001 in the preparation of drugs for preventing and treating colorectal cancer.

[0015] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] 1) Preventive oral administration of Lactobacillus mucosae LMSJ001 significantly reduced the size and weight of MC38 subcutaneous tumors;

[0017] 2) Lactobacillus mucosae 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 object of the present invention is to provide the use of equol, a metabolite of Lactobacillus mucosa LMSJ001, in the preparation of immunotherapy drugs for sensitizing microsatellite stable (immunotherapy-insensitive) colorectal cancer.

[0020] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0021] 1) Lactobacillus mucosa LMSJ001 can transform and produce the metabolite equol;

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

[0023] 3) Equol significantly enhances the efficacy of PD-1 antibody against CT26 subcutaneous tumors;

[0024] The fifth object of the present invention is to provide the use of equol, a metabolite of Lactobacillus mucosae LMSJ001, in the preparation of a drug for treating colorectal cancer.

[0025] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

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

[0028] 3) Equol significantly promoted apoptosis of HCT116 cells;

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

[0030] 5) Equol can enter HCT116 cells and exert tumor suppressive effects.

[0031] The sixth object of the present invention is to provide the use of equol, a metabolite of Lactobacillus mucosa LMSJ001, in the preparation of a drug that promotes ferroptosis in colorectal cancer.

[0032] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

[0034] 2) Equol significantly reduced the mitochondrial membrane potential level of HCT116 cells;

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

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

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

[0038] The seventh object of the present invention is to provide a PD-1 inhibitor sensitizer, a preparation containing mucosal Lactobacillus LMSJ001 and / or equol, thereby sensitizing the immunotherapy of microsatellite-stable colorectal cancer.

[0039] As a preferred technical solution of the present invention: the drug or sensitizer includes Lactobacillus mucosa LMSJ001 or its metabolite equol, a drug carrier and / or a pharmaceutically acceptable excipient.

[0040] As a preferred technical solution 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] The present invention provides the use of Lactobacillus mucosa LMSJ001 or its metabolite equol in the preparation of products related to the prevention of colorectal cancer and / or sensitization of colorectal cancer immunotherapy, which has at least the following advantages and beneficial effects:

[0042] 1) Lactobacillus mucosa LMSJ001 or its metabolite equol sensitizes microsatellite stable (immunotherapy-resistant) colorectal cancer to immunotherapy. Currently, there are no relevant literature or patent reports. This provides a new method for enhancing the efficacy of tumor immunotherapy.

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

[0044] 3) Equol significantly inhibits colorectal cancer, promotes tumor cell apoptosis, inhibits cell cycle, and promotes tumor cell ferroptosis, providing new clues and new ideas for future exploration of methods to inhibit tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1The whole genome sequencing map of Lactobacillus mucosae LMSJ001 and the whole genome comparison diagram with known Lactobacillus mucosae strains whose whole genome sequences have been published by NCBI; in the figure: A is the genome circle map of Lactobacillus mucosae LMSJ001; B is the average nucleotide similarity analysis diagram of Lactobacillus mucosae LMSJ001 and known Lactobacillus mucosae strains whose whole genome sequences have been published by NCBI.

[0046] Figure 2 This is a graphical representation of the immunotherapy effect of Lactobacillus mucosae LMSJ001 on sensitizing microsatellite stable (immunotherapy-resistant) colorectal cancer; in the figure: A is a schematic diagram of the tumor size of mice with CT26 subcutaneous tumors treated with Lactobacillus mucosae LMSJ001 and PD-1 antibody alone and in combination; B is a graph of the tumor growth curve of mice with CT26 subcutaneous tumors treated with Lactobacillus mucosae LMSJ001 and PD-1 antibody alone and in combination.

[0047] Figure 3 This is a graphical representation of the effect of preventive oral administration of Lactobacillus mucosa LMSJ001 in significantly inhibiting tumor growth in the MC38 subcutaneous tumor mouse model; in the figure: A is a schematic diagram of the model; 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 statistical graph of the abundance of Lactobacillus mucosa LMSJ001 in mouse feces.

[0048] Figure 4 The figure shows the significant inhibition of tumor growth in the MC38 subcutaneous tumor mouse model by Lactobacillus mucosa LMSJ001. In the figure: A is a schematic diagram of the model; B is a graph of tumor growth; C is a schematic diagram of tumor size; D is a statistical graph of tumor weight; E is a graph of tumor infiltrating CD8 + T cell ratio statistics.

[0049] Figure 5 Figure 3 is a diagram of the metabolomics analysis and mass spectrometry detection of the metabolite equol produced by Lactobacillus mucosae LMSJ001; in the figure: A is a volcano plot of differential metabolites in mouse serum after oral gavage with Lactobacillus mucosae LMSJ001 compared with the control group; B is a calorimetric plot (left) and VIP value plot (right) of differential metabolite expression in mouse serum after oral gavage with Lactobacillus mucosae LMSJ001 compared with the control group; C is the equol content in mouse feces in the Lactobacillus mucosae LMSJ001 gavage group and the control group; D is the equol content in the culture supernatant and blank culture medium after Lactobacillus mucosae LMSJ001 was cultured in vitro in M9 medium supplemented with daidzein (DZN).

[0050] Figure 6This is a graphical representation of the immunotherapy effect of equol-sensitized microsatellite-stable (immunotherapy-insensitized) colorectal cancer; in the figure: A is a schematic diagram of the tumor size of mice with CT26 subcutaneous tumors treated with equol and PD-1 antibodies alone and in combination; B is a graph of the tumor growth curve of mice with CT26 subcutaneous tumors treated with equol and PD-1 antibodies alone and in combination.

[0051] Figure 7 The figures are a graphic representation of the significant inhibitory effect of equol on colon cancer; in the figure, A is a graph showing the tumor size of mice with MC38 subcutaneous tumors significantly inhibited by equol intervention; B is a graph showing the tumor growth curve of mice with MC38 subcutaneous tumors significantly inhibited by equol intervention; C is a statistical graph showing the tumor weight of mice with MC38 subcutaneous tumors significantly inhibited by equol intervention; D is a statistical graph showing the proliferation of HCT116 and HT29 cells after intervention with different concentrations of equol detected by CCK8 assay; E is a graph showing the colony formation of HCT116 and HT29 cells after intervention with equol detected by plate cloning assay; F is a statistical graph showing the number of colony formation of HCT116 and HT29 cells after intervention with equol detected by plate cloning assay; G is a statistical graph showing the apoptosis rate of HCT116 cells after intervention with equol; H is a statistical graph showing the cell cycle of HCT116 cells after intervention with equol; I is a graph showing 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 Graph showing the effect of equol in promoting ferroptosis in colorectal cancer; in the figure: A is a statistical graph showing the reactive oxygen species level in HCT116 cells after equol intervention; B is a statistical graph showing the mitochondrial membrane potential level in HCT116 cells after equol intervention; C is a statistical graph showing the cell doubling number in HCT116 cells after equol intervention under the action of the ferroptosis inducer RSL3; D is a statistical graph showing the cell doubling number in HCT116 cells after equol intervention under the action of the ferroptosis inhibitor Fer-1; E is a statistical graph showing the lipid peroxide level in HCT116 cells after intervention with equol, equol and the ferroptosis inhibitor Fer-1. DETAILED DESCRIPTION

[0053] The technical solution of the present invention is 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, 2 g of fecal sample was collected from healthy elderly people in the longevity village of Lishui, Zhejiang Province. The sample was diluted with sterile saline and then spread on RCM medium plates. The plates were then anaerobically cultured at 37°C. Single colonies were selected for purification and culture.

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

[0057] (3) 16S rDNA sequencing identification: The LMSJ001 strain was amplified by colony PCR using universal bacterial primers and sent to Hangzhou Mingke Biological Company for 16S rDNA sequencing identification. Sequence alignment on NCBI showed that the length of the strain's 16S rDNA was 1405 bp, and the sequence was as follows. It was found that the sequence had the highest homology similarity with the 16S rDNA sequence of Limosilactobacillus mucosae (the alignment similarity was approximately 100%), and the strain was determined to be mucosal Lactobacillus.

[0058]

[0059]

[0060] (4) Whole genome identification: The whole genome of the above-mentioned Lactobacillus mucosa LMSJ001 was further sequenced at Shanghai Meiji Biotechnology Co., Ltd., and the sequencing results were subjected to Circos genome circle map analysis, evolutionary tree analysis and pan-genome Venn diagram analysis. The results are as follows: Figure 2 A, is the complete genome sequencing of LMSJ001 strain (Circos genome circle map analysis); Figure 2 As shown in Figure B, the ANI value is calculated by aligning homologous regions between genomes and calculating the average nucleotide identity, reflecting genomic similarity. Generally, genomes with an ANI value greater than 99.9% are considered identical strains. Lactobacillus mucosae LMSJ001 differs from other reported Lactobacillus mucosae strains. Based on these results and literature research, LMSJ001 is distinct from previously reported strains and is a newly discovered Lactobacillus mucosae strain.

[0061] This strain was deposited in the General Microbiology Center of the China Culture Collection Administration on February 26, 2024. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC NO.29885, and the recommended taxonomic name is Limosilactobacillus mucosae.

[0062] Example 2: Lactobacillus mucosae LMSJ001 enhances immunotherapy of microsatellite-stable (immunotherapy-insensitive) colorectal cancer

[0063] Six-week-old male Balb / c mice were randomly divided into four groups, with 10 mice in each group. The mice were allowed to drink water containing 0.2 mg / mL ampicillin, neomycin sulfate, metronidazole, and 0.1 mg / mL vancomycin freely for 3 days to initially eliminate intestinal flora. The mice were subcutaneously injected with a mixture of CT26 cells and Matrigel (cell to Matrigel ratio of 1:1) at a dose of 10 6 cells / mouse, and the injection volume was 100 μL / mouse. Four groups of mice were respectively given PBS, Lactobacillus mucosa LMSJ001, PBS+PD-1 antibody, and Lactobacillus mucosa LMSJ001+PD-1 antibody intervention. Among them, PBS and Lactobacillus mucosa LMSJ001 were administered by gavage at a dose of 1×10 9 CFU / mouse, gavage volume 200 μL / mouse, once daily; PD-1 antibody was administered via intraperitoneal injection at a dose of 100 μg / mouse, once on days 7, 10, and 13. During modeling, tumor size was measured every two days. After modeling, tumor size and weight were calculated.

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

[0065] Example 3: Preventive oral administration of Lactobacillus mucosa LMSJ001 significantly inhibits tumor growth in the MC38 subcutaneous tumor mouse model

[0066] Six-week-old male C57BL / 6 mice were randomly divided into two groups, with 10 mice in each group. Figure 3 As shown in A, mice were given 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 eliminate intestinal flora, which is conducive to the colonization of transplanted bacteria. After the intestinal flora was eliminated, the two groups were given PBS and Lactobacillus mucosae LMSJ001 by gavage, respectively, at a dose of 1×10 9 CFU / mouse, oral administration volume 200 μL / mouse, once a day. After 5 days of oral administration, mice were subcutaneously injected with a mixture of MC38 cells and matrigel (cell to matrigel ratio of 1:1) at a dose of 10 6 cells / mouse, with an injection volume of 100 μL / mouse, which was designated as Day 0 (DAY0). Oral gavage continued daily until Day 21. During modeling, tumor size was measured every two days. After modeling, tumor size and weight were calculated, and feces were collected for detection of changes in the abundance of Lactobacillus mucosa LMSJ001 in the feces using qPCR.

[0067] The results are as follows Figure 3The modeling diagram is shown in Figure A. Lactobacillus mucosae LMSJ001 can significantly reduce tumor growth size (B and C) and tumor weight (D) in the MC38 subcutaneous tumor mouse model. Compared with the control group, the abundance of Lactobacillus mucosae LMSJ001 in the feces of mice in the Lactobacillus mucosae LMSJ001 gavage group was significantly increased (E).

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

[0069] Six-week-old male C57BL / 6 mice were randomly divided into two groups, with 10 mice in each group. Figure 4 As shown in A, mice were given 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 eliminate intestinal flora and facilitate the colonization of transplanted bacteria. Mice were subcutaneously injected with a mixture of MC38 cells and matrigel (cell to matrigel ratio of 1:1) at a dose of 10 6 The two groups were given PBS and Lactobacillus mucosa LMSJ001 by gavage, with a dose of 1×10 9 CFU / mouse, gavage volume 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 counted, and the mouse tumor tissue was collected for flow cytometry to detect the proportion of CD8+ T cells in the tumor. In addition, the mouse serum and feces were collected for further analysis.

[0070] The results are as follows Figure 4 As shown in the figure, the modeling diagram is shown in A. Lactobacillus mucosa LMSJ001 can significantly reduce the tumor size (B and C) and tumor weight (D) of mice with MC38 subcutaneous tumors. 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 Produced by Lactobacillus mucosa LMSJ001

[0072] Metabolomics analysis was performed on the mouse serum collected in Example 3 to analyze differential metabolites in the mouse serum after oral administration of L. mucosae LMSJ001. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was performed on the mouse feces collected in Example 3 to analyze the equol content in the mouse feces of the L. mucosae LMSJ001 oral administration group and the control group. After in vitro cultivation of L. mucosae LMSJ001 in M9 medium supplemented with the equol precursor daidzein (DZN), the equol content in the culture supernatant and blank culture medium was measured 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 equol content in the mouse serum was significantly upregulated after oral gavage of Lactobacillus mucosae LMSJ001 (A and B); compared with the control group, the equol content in the mouse feces was significantly increased after oral gavage of Lactobacillus mucosae LMSJ001 (C); compared with the blank culture medium, the equol content in the in vitro culture fermentation broth of Lactobacillus mucosae LMSJ001 was significantly increased (D).

[0074] Example 6: Equol sensitizes microsatellite stable (immunotherapy-insensitive) colorectal cancer immunotherapy

[0075] Six-week-old male Balb / c mice were randomly divided into four groups, with 10 mice in each group. The mice were subcutaneously injected with a mixture of CT26 cells and matrigel (cell to matrigel ratio of 1:1) at a dose of 10 6 The four groups of mice were divided into a control group, an equol group, a control + PD-1 antibody group, and an equol + PD-1 antibody group. The control solvent and equol were administered by oral gavage. The intervention dose of equol was 10 mg / kg mouse body weight, and the oral volume was 200 μL / mouse. The control group mice were gavaged with an equal volume of sodium carboxymethylcellulose solution. The PD-1 antibody was administered by intraperitoneal injection at a dose of 100 μg / mouse, once on days 7, 10, and 13, respectively. During the modeling process, tumor size was measured every two days.

[0076] The results are as follows Figure 6 As shown in the results, equol treatment alone, especially in combination with PD-1 antibody, could significantly reduce the tumor size of mice bearing CT26 subcutaneous tumors.

[0077] Example 7: Equol significantly inhibits colon cancer

[0078] In the in vivo experiment, 6-week-old male C57BL / 6 mice were randomly divided into two groups, with 10 mice in each group. The mice were subcutaneously injected with a mixture of MC38 cells and matrigel (cell to matrigel ratio of 1:1) at a dose of 10 6The two groups were gavaged with a control solvent and equol, respectively. The equol intervention dose was 10 mg / kg mouse body weight, with a gavage volume of 200 μL / mouse. The control group received an equal volume of sodium carboxymethylcellulose solution by gavage once daily until day 21. During modeling, tumor size was measured every two days. After modeling, tumor size and weight were calculated.

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

[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 well. Cultures were continued for 5 days. The culture medium was aspirated daily from one plate, and 100 μL of complete 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 the cells adhered, control solvent and 200 μM equol were added, respectively, with at least 3 replicates per group. After the cells grew clonal spots, they were fixed with 4% paraformaldehyde solution for 30 minutes, then stained with 0.1% crystal violet solution for 20 minutes. After being washed with PBS, the plates were air-dried in a clean bench, photographed with a digital camera, and the clonal spots were counted and analyzed using imageJ software to reflect the proliferation potential of individual cells.

[0082] For cell apoptosis detection, cells were seeded in 6-well plates with 10 5 After the cells adhered, control solvent and 200 μM equol were added, with at least three replicates per group. After 48 hours of culture, the cells were harvested and apoptosis was detected using the Annexin V-FITC / PI double staining method. The specific steps were as follows: wash the cells twice with pre-chilled PBS, resuspend the cells in binding buffer, then add Annexin V-FITC and PI staining solution, and incubate in the dark for 15 minutes. Finally, the cell 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 at a rate of 10 5After cells adhered, control solvent and 200 μM equol were added, with at least three replicates per group. After 48 hours of culture, cells were harvested, washed twice with pre-chilled PBS, fixed with 70% ethanol, and incubated overnight at 4°C. After fixation, cells were washed with PBS and PI staining solution containing RNase A was added, incubating in the dark for 30 minutes. Finally, cell cycle distribution was assessed by flow cytometry, analyzing the proportions of cells in the G0 / G1, S, and G2 / M phases to evaluate the effects of equol on cell cycle progression.

[0084] For subcellular fluorescence localization detection, cells were seeded on confocal microplates. After the cells adhered, CY5-labeled equol 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 fluorescent-labeled cell membrane Dio probe was added and incubated at 37°C in the dark for 20 minutes. After washing with PBS, Hoechst 33342 staining solution was added and incubated at 37°C for 10 minutes to label the cell nuclei. The cells were observed and images were collected under a laser confocal microscope.

[0085] The results are as follows Figure 7 As shown, compared with the control group, equol treatment significantly reduced tumor size (A and B) and tumor weight (C) in mice bearing MC38 subcutaneous tumors. Compared with the control group, equol treatment significantly inhibited the proliferation (D) and clonogenicity (E and F) of colon cancer cells HCT116 and HT29, significantly promoted apoptosis of HCT116 cells (G), 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: Equol promotes ferroptosis in colorectal cancer

[0087] Human colon cancer cells HCT116 were revived using McCoy's 5A medium supplemented with 10% fetal bovine serum as complete medium and cultured in a 37°C, 5% CO2 incubator. For the detection of cellular reactive oxygen species levels, cells were seeded in 6-well plates with 10 cells per well. 5 After the cells adhered to the wall, control solvent and 200 μM equol were added, with at least 3 replicates per group. After 48 hours of culture, the 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. Washed 3 times with PBS, the green fluorescence intensity was detected by flow cytometry (excitation / emission wavelength: 488 / 525 nm), the ROS level was quantified, and the data were analyzed by FlowJo software. For the mitochondrial membrane potential level detection, cells were seeded in 6-well plates, with 10 cells seeded per well. 5After cells adhered, control solvent and 200 μM equol were added, with at least three replicates per group. After 48 hours of culture, cells were harvested, JC-1 staining solution (5 μg / mL) was added, and 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 measured by flow cytometry, and the proportion of cells with decreased mitochondrial membrane potential was calculated. For analysis of cell doublings in response to the ferroptosis inducer (RSL3) combined with equol, cells were seeded in 96-well plates, with 1000 cells per well. After cells adhered, they were divided into three groups, each receiving 0 μM, 0.5 μM, or 1 μM of the ferroptosis inducer RSL3. Each group was further divided into two groups, each receiving control solvent and 200 μM equol, respectively. A blank control group containing only control solvent was also set up, with at least three replicates per group. On the 0th and 4th day 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 value at 450 nm (OD450nm) was detected. The calculation formula is: cell doubling number = log2 (N / N0), where N is the current cell number and N0 is the initial cell number. For the cell doubling number analysis of ferroptosis inhibitor (Fer-1) combined with equol, the grouping settings were the same as above, RSL3 was replaced with ferroptosis inhibitor Fer-1 (0 μM, 0.5 μM, 1 μM), and the rest of the 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 After cells adhered, they were divided into the following experimental groups: control group, equol group (200 μM), and equol + Fer-1 group (200 μM equol + 1 μM Fer-1). At least three replicates were set up for each group. After 48 hours of treatment, cells were harvested and C11-BODIPY 581 / 591 probe (2 μM) was added. The cells were incubated at 37°C in the dark for 30 minutes. After washing with PBS, 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 was measured by flow cytometry to quantify lipid peroxidation levels.

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

[0089] The above-mentioned specific implementation methods are used to illustrate the present invention and are only preferred embodiments of the present invention, rather than limiting the present invention. Any modifications, equivalent substitutions, improvements, etc. made to the present invention within the spirit of the present invention and the scope of protection of the claims shall fall within the scope of protection of the present invention.

Claims

1. Application of mucosal lactobacilli in the preparation of sensitized immunotherapy drugs for microsatellite-stable colorectal cancer.

2. The use according to claim 1, characterized in that: The mucosal lactobacillus is Limosilactobacillus mucosae LMSJ001, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC NO.29885.

3. Application of mucosal lactobacillus in the preparation of drugs for preventing and treating colorectal cancer.

4. Application of equol in the preparation of sensitizing immunotherapy drugs for microsatellite-stable colorectal cancer.

5. Application of equol in the preparation of drugs for the treatment of colorectal cancer.

6. Application of equol in the preparation of drugs that promote ferroptosis in colorectal cancer.

7. A PD-1 inhibitor sensitizer, characterized in that Preparations containing Lactobacillus mucosae and / or equol to sensitize microsatellite-stable colorectal cancer to immunotherapy.

8. The use according to any one of claims 1 to 6 or the sensitizer according to claim 7, characterized in that The dosage form of the drug or the sensitizer is pills, tablets, powders, capsules, granules, suspensions, injections, oral solutions, enemas or tube feeding preparations.

9. The use according to any one of claims 1 to 6 or the sensitizer according to claim 7, characterized in that It includes pharmaceutical carriers and / or pharmaceutically acceptable excipients.

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