Application of paraclostridium graciliatum in preparation of medicine for treating colorectal cancer

Abstract

The invention provides an application of paraclostridium graciliatum in preparation of a medicine for treating colorectal cancer. According to the present invention, the culture supernatant of the Paracostridium tenue CGMCC No.31232 can effectively inhibit the proliferation, the clone formation and the migration of the colorectal cancer cells, promote the apoptosis of the colorectal cancer cells, and induce the cell cycle arrest; a colorectal cancer transplanted tumor mouse model verifies that the strain can inhibit tumor formation of a colorectal cancer transplanted tumor mouse, regulate intestinal microbiota, influence the expression of immune factors in tumors and generate metabolites with an anti-CRC effect, has the effect of improving the colorectal cancer of the mouse, has no obvious toxic or side effect on animals, and can be used for preparing a medicine for treating the colorectal cancer of the mouse. The invention provides a new strategy for treatment of colorectal cancer, and has a potential clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to application of Paraclostridium gracilis in preparing medicine for treating colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract that often occurs at the junction of the rectum and sigmoid colon. It is mainly caused by abnormal proliferation of intestinal mucosal epithelial cells. At the same time, tumor cells have the ability to invade and spread, posing a serious threat to human health. Therefore, improving the prevention and treatment measures of CRC is particularly important.

[0003] Currently, the treatment of CRC is mainly surgery, supplemented by chemotherapy and radiotherapy. However, the recurrence rate of colorectal cancer after surgery is high, and radiotherapy and chemotherapy have significant side effects. Cancer cells are prone to developing resistance to chemotherapy drugs, resulting in limited chemotherapy effectiveness. Therefore, it is urgent to develop new treatment options that are safer, milder, and have fewer side effects, such as the development of probiotics, prebiotics, and other microecological preparations for adjuvant therapy. In recent years, more and more experimental studies and clinical trials have shown that probiotics have the effect of preventing or alleviating CRC. However, the inhibitory effects and mechanisms of different types of probiotics on CRC are different and need further study.

[0004] Paraclostridium gracilis ( Paraclostridium tenue ) Formerly known as Eubacterium gracilis ( Eubacterium tenue ), and was later classified into the genus Paraclostridium in 2024. Eubacterium is an important intestinal bacteria in the colon of healthy people and is part of the core intestinal flora of the human body. Some Eubacterium strains (such as Eubacterium hallii ( Eubacterium hallii ) and Eubacterium mucilaginosum ( Eubacterium limosum ) has been recognized as a potential beneficial microorganism and a candidate for the next generation of probiotics. Clostridium tenuis is a significant representative of the human gut microbiota and can be isolated from human fecal samples or dairy products. However, there are currently no reports on the biological characteristics of Clostridium tenuis and its effects on improving CRC. Summary of the Invention

[0005] The present invention provides use of Paraclostridium gracilis in preparing a medicine for treating colorectal cancer.

[0006] In the present invention, Paraclostridium gracilis ( Paraclostridium tenue ) Pt517 was deposited on July 3, 2024, at the General Microbiology Center of China Culture Collection Administration (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), and was named Paraclostridium gracilis. Paraclostridium tenue, the deposit number is CGMCC No.31232, and the strain has been disclosed in patent application CN119752719A.

[0007] Paraclostridium gracilis ( Paraclostridium tenue ) Pt517 is a Clostridium parahaemolyticus with probiotic properties that was isolated and purified from healthy human feces. The present invention has experimentally demonstrated that the strain is harmless to animals and has high safety. Cell experiments and animal experiments have also verified that the strain has the efficacy of treating colorectal cancer. The culture supernatant of the strain can effectively inhibit the proliferation, cloning and migration of colorectal cancer cells, promote apoptosis of colorectal cancer cells, and induce cell cycle arrest. The strain can significantly reduce the tumor volume and weight of colorectal cancer-bearing mice, downregulate the expression of Ki-67 in tumors, and inhibit the proliferation of colorectal cancer cells in mice. In addition, the strain can also regulate the intestinal microbiota, affect the expression of immune factors in tumors, and produce metabolites with anti-CRC effects.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a Paraclostridium tenue ) Application of Pt517 in preparing a product for improving or treating colorectal cancer; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0010] In a second aspect, the present invention provides a Paraclostridium tenue ) Application of Pt517 in the preparation of a product for preventing colorectal cancer; the paraclostridium slenderum ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0011] In a third aspect, the present invention provides a Paraclostridium tenue ) Application of Pt517 in the preparation of a product for inhibiting proliferation, cloning and / or migration of colorectal cancer cells; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0012] In a fourth aspect, the present invention provides Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for promoting apoptosis of colorectal cancer cells; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0013] In a fifth aspect, the present invention provides Paraclostridium gracilis ( Paraclostridium tenue) Application of Pt517 in the preparation of a product for blocking the colorectal cancer cell cycle; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0014] The present invention found that Clostridium gracilis ( Paraclostridium tenue ) Pt517 can arrest the cell cycle of colorectal cancer cells at the G0 / G1 phase, preventing them from entering the S phase for DNA synthesis. Cell growth and division will be stopped, thereby affecting the normal proliferation of tumor cells.

[0015] In a sixth aspect, the present invention provides Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for regulating the secretion of immune factors in patients with colorectal cancer; the Paraclostridium tenuis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0016] Preferably, the immune factors include anti-tumor immune factors and immunosuppressive factors; wherein, for anti-tumor immune factors (such as TNF-α, IFN-γ, CD8), the regulation is to increase the content of the anti-tumor immune factors, and for immunosuppressive factors (such as IL-6, IL-10, TGF-β), the regulation is to reduce the content of the immunosuppressive factors.

[0017] Preferably, the immune factor is an immune factor in a tumor.

[0018] In a seventh aspect, the present invention provides Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for regulating the intestinal flora of patients with colorectal cancer; the Paraclostridium tenuis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0019] Preferably, the intestinal flora is regulated by increasing the number of beneficial bacteria, such as Parabacteroides archaeopteris ( Parabacteroides goldsteinii )、Lachnospiraceae bacteria G11( Lachnospiraceae bacteria G11), cecal Enterobacter B7 ( Enterorhabdus caecimuris B7), and reduced the relative abundance of potential pathogens, such as the Christensenaceae R7 group bacteria ( Christensenellaceae The relative abundance of R7 group) makes the intestinal flora composition structure of colorectal cancer patients closer to normal.

[0020] In an eighth aspect, the present invention provides Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of products for increasing the content of palmitic acid and / or stearic acid; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

[0021] Paraclostridium gracilis ( Paraclostridium tenue ) Pt517 has the function of secreting palmitic acid and stearic acid, so it can increase the content of palmitic acid and / or stearic acid.

[0022] In the above application, the product is preferably a medicine.

[0023] In the above application, the active ingredient of the product includes the paraclostridium gracilis ( Paraclostridium tenue ) Pt517 or a culture thereof.

[0024] Preferably, the culture comprises Paraclostridium gracilis ( Paraclostridium tenue ) Pt517 and / or its culture supernatant.

[0025] Preferably, the culture is a culture of Clostridium gracilis ( Paraclostridium tenue ) Pt517 is obtained by anaerobically culturing in a reinforced medium for Clostridia (RCM), wherein the culture temperature is preferably 35-40°C.

[0026] Preferably, the product is a capsule, freeze-dried powder or bacterial liquid preparation.

[0027] The beneficial effects of the present invention include at least: the paraclostridium gracilis provided by the present invention ( Paraclostridium tenue The culture supernatant of CGMCC No. 31232 can effectively inhibit the proliferation, cloning, and migration of colorectal cancer cells, promote apoptosis, and induce cell cycle arrest. Validated in a colorectal cancer xenograft mouse model, this strain can inhibit tumor formation in mice bearing colorectal cancer xenografts, regulate the intestinal microbiome, influence the expression of immune factors in tumors, and produce metabolites with anti-CRC effects. This strain has the potential to improve colorectal cancer in mice, with no significant toxic side effects and a high safety profile. This provides a new strategy for the treatment of colorectal cancer and has broad application value in pharmaceuticals and various forms of probiotic products, with potential clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is the effect of the culture supernatant of the Clostridium gracilis CGMCC No. 31232 strain in Example 1 of the present invention on the proliferation of colorectal cancer cells.

[0030] Figure 2 This is the effect of the culture supernatant of the Clostridium gracilis CGMCC No. 31232 strain in Example 1 of the present invention on the colony formation of colorectal cancer cells.

[0031] Figure 3 This is the effect of the culture supernatant of the Clostridium gracilis CGMCC No. 31232 strain in Example 1 of the present invention on the migration of colorectal cancer cells.

[0032] Figure 4 This is the effect of the culture supernatant of the Clostridium gracilis CGMCC No. 31232 strain in Example 1 of the present invention on the apoptosis of colorectal cancer cells.

[0033] Figure 5 This is the effect of the culture supernatant of the Clostridium gracilis CGMCC No. 31232 strain in Example 1 of the present invention on the cell cycle of colorectal cancer cells.

[0034] Figure 6 This is the effect of the Clostridium gracilis CGMCC No. 31232 strain in Example 2 of the present invention on tumor formation in mice bearing colorectal cancer transplanted tumors.

[0035] Figure 7 This is the effect of the Clostridium gracilis CGMCC No. 31232 strain in Example 2 of the present invention on the pathological changes and Ki-67 staining results of tumor tissues in mice with colorectal cancer transplanted tumors.

[0036] Figure 8 This is the effect of the Clostridium gracilis CGMCC No. 31232 strain in Example 2 of the present invention on the content of immune factors in tumor tissues of mice with colorectal cancer transplanted tumors.

[0037] Figure 9 This is the effect of the Clostridium gracilis CGMCC No. 31232 strain in Example 2 of the present invention on the intestinal flora of mice bearing colorectal cancer transplanted tumors.

[0038] Figure 10 This is the effect of the Clostridium gracilis CGMCC No. 31232 strain in Example 2 of the present invention on the serum metabolomics of mice bearing colorectal cancer transplanted tumors.

[0039] Figure 1-5 In the table, Control represents the enhanced Clostridium culture medium (blank control), EcCS represents the culture supernatant of Escherichia coli (negative control), and Pt517CS represents the culture supernatant of Paraclostridium tenuis CGMCC No. 31232. Figure 6-10 In the table, NC represents the blank control group, PBS represents the colorectal cancer model group, and Pt517 represents the intervention group with Clostridium truncatum CGMCC No. 31232. N = 4-6. Results are presented as x ± SEM; *: P < 0.05; **: P < 0.01; ***: P < 0.001. DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0041] Example 1 Effect of the culture supernatant of Clostridium gracilis CGMCC No. 31232 on colorectal cancer cells 1. Subculture of Clostridium gracilis Remove a vial of Clostridium paratenicum CGMCC No. 31232 strain frozen at -80°C and slowly thaw. Use a sterile inoculating loop to evenly spread an appropriate amount of the preservation solution onto an RCM solid plate. Incubate at 37°C under anaerobic conditions for 48 hours. After 48 hours, remove the plate and use a sterile inoculating loop to pick a single white, translucent colony and transfer it to fresh culture medium for subculture.

[0042] 2. Preparation of Paraclostridium gracilis culture supernatant The strains of Clostridium truncatum were serially passaged three times on the solid medium of RCM.

[0043] Reinforced Medium for Clostridia (RCM) was sterilized by autoclaving at 121°C for 15 min and then cooled to room temperature for later use.

[0044] Add 48 mL of sterilized RCM to a 50 mL centrifuge tube and 2 mL of sterilized RCM to a turbidimetric tube. Use an inoculation loop to pick up a certain amount of Clostridium subtilisinus CGMCC No. 31232 and place it in each tube. Adjust the turbidity of the bacterial suspension to 4.0 with RCM. After mixing, add 2 mL of the bacterial suspension to the 50 mL centrifuge tube.

[0045] The centrifuge tube was placed in an anaerobic chamber and incubated at 37 °C for 48 h.

[0046] After 48 h, the bacterial solution in the anaerobic chamber was removed, centrifuged at 3800 rpm for 10 min, and the supernatant was collected.

[0047] The supernatant was filtered into a new centrifuge tube using a 10 mL syringe and a 0.22 μM filter membrane. The obtained bacterial culture supernatant (i.e., Pt517CS) was frozen in a -40 °C refrigerator for later use.

[0048] 3. Inhibitory effect of culture supernatant of Clostridium gracilis on the proliferation of colorectal cancer cells Cell culture: CT26, HT29, and NCM460 cells were seeded in RPMI-1640 medium supplemented with 10% fetal bovine serum, and Caco2 cells were seeded in RPMI-1640 medium supplemented with 20% fetal bovine serum. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 h.

[0049] Cell proliferation ability detection (MTT method): (1) Plating: Digest the cells in the logarithmic growth phase with 0.25% trypsin to remove them from the bottom of the cell bottle, and prepare a cell suspension in RPMI-1640 culture medium containing 10% fetal bovine serum. Adjust the cell concentration to 5×10 4 cells / mL, inoculated into a 96-well plate, 100 μL per well, and then incubated the 96-well plate in a 37°C, 5% CO2 incubator overnight.

[0050] (2) Sample addition: When the cell density reaches 80%~90%, aspirate the liquid in the 96-well plate and add RCM (blank control), Escherichia coli MG1655 culture supernatant (EcCS, negative control) or Paraclostridium gracilis culture supernatant (Pt517CS) according to the experimental design, 100 μL per well, and then place the 96-well plate in an incubator and culture for 48 h.

[0051] (3) Add MTT: After 48 h, discard the liquid in the wells, add 100 μL of MTT solution to each well, and culture in an incubator for 4 h.

[0052] (4) Add Formazan solution: Aspirate the solution in the wells, add 110 μL Formazan solution to each well, and place the 96-well plate on a shaker and shake at low speed for 10 minutes to fully dissolve the crystals.

[0053] (5) Result determination: Use an enzyme-labeled instrument to measure the absorbance of each well at a wavelength of 490 nm.

[0054] (6) Calculation results: Cell proliferation inhibition rate = (1-OD value of treatment group / OD value of control group) × 100%. Four parallel experiments were performed and the experimental results were statistically analyzed using GraphPad software.

[0055] Results: The effects of Pt517CS on the proliferation of colorectal cancer cells were as follows: Figure 1As shown. Among them, Figure 1 Figure A is a comparison of the inhibition of Pt517CS on the proliferation of different cells. It can be seen that Pt517CS has a strong inhibitory effect on the proliferation of three colorectal cancer cells, CT-26, HT-29 and Caco-2, and the cell proliferation inhibition rate is significantly higher than that of EcCS (P<0.05); while it has a weak inhibitory effect on the proliferation of NCM460 (human normal colon epithelial cells), and there is no statistical difference in the cell proliferation inhibition rate with EcCS. Figure 1 Figures B and C are comparative diagrams of the inhibition of CT-26 and HT-29 cell proliferation by Pt517CS at different concentration gradients. It can be seen that the inhibition of Pt517CS on the proliferation of the two colorectal cancer cells is concentration-dependent. Figure 1 Based on the experimental results of B and C, 14% Pt517CS (formula: 14% Pt517CS + 10% fetal bovine serum + 76% RPMI-1640 culture medium) was selected for subsequent experiments.

[0056] 4. Inhibitory effect of culture supernatant of Clostridium gracilis on colony formation of colorectal cancer cells Clone formation assay method: (1) Plating: After digesting, passaged, harvesting, and counting CT-26 cells, 5000 cells / well were seeded into 6-well plates and incubated in an incubator for 24 h.

[0057] (2) Sample addition: Prepare a mixture of 14% RCM (blank control) / EcCS (negative control) / Pt517CS+10% fetal bovine serum+76% RPMI-1640 culture medium, aspirate the liquid in the 6-well plate, add 1 mL of the mixture to each well, place the plate in a cell culture incubator and continue culturing. Observe the growth of cells in the 6-well plate every day and change the medium every 3 days.

[0058] (3) Fixation: After the cells in the control group in the 6-well plate are fully grown, remove the 6-well plate, discard the liquid in the wells, gently rinse the cells 1-2 times with PBS solution, add 1.5 mL of 4% paraformaldehyde to each well, and fix at room temperature for 30 min.

[0059] (4) Staining: Aspirate the polymethanol in the wells and gently rinse the cells 1-2 times with PBS. Add 1 mL of 10% Giemsa stain to each well and stain for 15 minutes. Then discard the liquid in the wells and gently wash the cells with running water to remove excess stain.

[0060] (5) Photographing and counting: After the 6-well plate is dried, take a photo and save it. Count the number of clones formed in each well. Perform three parallel experiments and use GraphPad software to statistically analyze the experimental results.

[0061] Results: The effect of Pt517CS on the colony formation of colorectal cancer cells was as follows: Figure 2 As shown. Among them, Figure 2 A is a representative picture of cell clone formation after CT-26 cells were treated with various methods. Figure 2 Figure B is a statistical comparison of the number of cell clones formed after CT-26 was treated with different methods. It can be seen that compared with RCM and EcCS, Pt517CS can significantly reduce the number of CT-26 cell clones (P<0.05), indicating that it can inhibit the proliferation and clone formation of colorectal cancer cells.

[0062] 5. Inhibitory effect of culture supernatant of Clostridium gracilis on the migration of colorectal cancer cells Colorectal cancer cell migration assay method: (1) Plating: After digesting, passage, collecting, and counting CT-26 cells, the cell concentration was adjusted to 1×10 5 cells / mL, add 500 μL of cell suspension to each well of a 24-well plate, place in a cell culture incubator, and culture overnight.

[0063] (2) Scratching: When the cell density reaches 80%-90%, use the tip of a 1000 μL pipette to draw a straight line perpendicular to the bottom surface of the 24-well plate, keeping the scratch width consistent.

[0064] (3) Sample addition: Prepare a mixture of 14% RCM (blank control) / EcCS (negative control) / Pt517CS + 10% fetal bovine serum + 76% RPMI-1640 culture medium, discard the cell culture medium in the 24-well plate, gently rinse with PBS 1-2 times, then add 500 μL of the mixture to each well and place in the incubator for continued culture.

[0065] (4) Taking photos: Observe each well under a microscope and take photos at 0 h, 24 h, and 48 h after adding the sample.

[0066] (5) Statistics: Three parallel experiments were performed. The scratch area was measured using Image J software, and the cell migration rate was calculated: cell migration rate (%) = (0 h scratch area - 48 h or 24 h scratch area) / 0 h scratch area × 100%. The experimental results were statistically analyzed using GraphPad software.

[0067] Results: The cell scratch method was used to evaluate the effect of Pt517CS on CT-26 cell migration. Figure 3 As shown. Among them, Figure 3 A is a representative picture of cell migration after CT-26 cells were treated with various methods, and the gray shaded area indicates the area of ​​cell growth. Figure 3 B is a statistical comparison of cell migration rates after CT-26 cells were treated with different methods. Figure 3It can be seen that compared with RCM and EcCS, Pt517CS can significantly inhibit the migration of CT-26 cells.

[0068] 6. Promoting effect of culture supernatant of Clostridium gracilis on apoptosis of colorectal cancer cells Colorectal cancer cell apoptosis experimental method: (1) Plating: CT-26 cells were digested, passaged, collected, and counted, and 2×10 cells were plated per well. 5 Cells were plated in 6-well plates and incubated in an incubator overnight.

[0069] (2) Sample addition: After the cells are well attached, prepare a mixture of 14% RCM (blank control) / EcCS (negative control) / Pt517CS + 10% fetal bovine serum + 76% RPMI-1640 culture medium. Discard the culture medium in the 6-well plate and add 1.5 mL of the mixture to each well. Place the plate in a cell culture incubator and continue culturing for 24 h or 48 h.

[0070] (3) Collect cells: Collect the cell culture medium into a new centrifuge tube. Add 1 mL of EDTA-free trypsin to each well. Immediately remove the trypsin after wetting the cells. Then, add 1 mL of culture medium containing 10% fetal bovine serum to each well. Gently pipette to detach the cells. Collect the cells of the same group into the same centrifuge tube. Centrifuge at 1000 rpm for 5 min at 4°C and discard the supernatant.

[0071] (4) Washing: Add 1 mL of ice-cold PBS solution and gently pipette to suspend the cells. Transfer the resuspended liquid to a new 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at 4 °C. Discard the supernatant.

[0072] (5) Staining: Add 100 μL of diluted 1× binding buffer to each sample tube to resuspend the cells, then add 5 μL of Annexin V-FITC (stock concentration is 20 μg / mL), mix gently, and stain at room temperature in the dark for 15 min. According to the number of samples, prepare a mixture according to the volume ratio of PI: 1× Binding buffer = 1:79. Add 400 μL of the mixture to each tube of sample before loading, and stain at room temperature in the dark for 5 min.

[0073] (6) On-machine detection: The cell suspension was thoroughly blown to disperse it, and the cell suspension was filtered through a 40 μM cell sieve into a flow cytometer. The flow cytometer was used for detection. Three parallel experiments were performed, and data were analyzed using FlowJo and GraphPad software.

[0074] Results: CT-26 cell lines were stimulated with RCM or Pt517CS for 48 h and then the apoptosis of tumor cells was detected by PI / Annexin V-FITC double staining. Figure 4 shown. Figure 4 A is a scatter plot of cell apoptosis after CT-26 cells were treated with various methods. The upper left quadrant of the scatter plot represents mechanically damaged cells, the upper right quadrant represents late apoptotic or secondary death cells, the lower left quadrant represents normal cells, and the lower right quadrant represents early apoptotic cells. Figure 4 B is a statistical comparison of cell apoptosis after CT-26 cells were treated with different methods. Figure 4 It can be seen that compared with RCM, Pt517CS can significantly increase the proportion of early apoptosis, late apoptosis and total apoptotic cells in CT-26 cells, suggesting that its inhibitory effect on CT-26 cell proliferation may be achieved by inducing cell apoptosis.

[0075] 7. Inhibitory effect of culture supernatant of Clostridium gracilis on colorectal cancer cell cycle Experimental methods for arresting the cell cycle of colorectal cancer cells: (1) Plating: CT-26 cells were digested, passaged, collected, and counted, and 1×10 cells were plated per well. 6 Cells were plated in 6-well plates and incubated in an incubator overnight.

[0076] (2) Sample addition: Prepare a mixture of 14% RCM (blank control) / EcCS (negative control) / Pt517CS + 10% fetal bovine serum + 76% RPMI-1640 culture medium. After the cells are well attached, discard the culture medium in the 6-well plate and add 1.5 mL of the mixture to each well. Place the cells in a cell culture incubator and continue culturing for 48 h.

[0077] (3) Cell collection: After 48 h, discard the liquid in the wells and add 1 mL of EDTA-free trypsin to each well. Immediately remove the trypsin after wetting the cells. Then, add 1 mL of culture medium containing 10% fetal bovine serum to each well. Gently pipette to detach the cells. Collect the cells of the same group in the same centrifuge tube. Centrifuge at 1000 rpm for 10 min at 4°C and discard the supernatant.

[0078] (4) Washing: Add 5 mL of ice-cold PBS solution, gently blow to suspend the cells, centrifuge at 1000 rpm at 4°C for 10 min, and discard the supernatant.

[0079] (5) Fixation: Prepare 70% ethanol, cool in an ice bath, add 5 mL of pre-cooled ethanol while shaking, and fix at 4°C overnight.

[0080] (6) Washing: Centrifuge at 1000 rpm at 4°C for 15 min, discard the supernatant. Add 1 mL of PBS to resuspend the cells, transfer the cell suspension to a new 1.5 mL centrifuge tube, label it, centrifuge at 1000 rpm at 4°C for 15 min, discard the supernatant, and add 100 μL of PBS to resuspend the cells.

[0081] (7) Staining: Before loading the tube, add 500 μL of Pl / RNase staining buffer to each centrifuge tube, mix well, and stain at room temperature in the dark for 15 min.

[0082] (8) On-machine detection: The cell suspension was thoroughly blown to disperse it, and the cell suspension was filtered through a 40 μM cell sieve into a flow cytometer. The flow cytometer was used for detection. Four parallel experiments were performed, and data were analyzed using ModfitLT 5 and GraphPad software.

[0083] Results: CT-26 cell lines were stimulated with RCM or Pt517CS, and the cell cycle distribution of tumor cells was detected by PI single staining after 48 hours. Figure 5 shown. Figure 5 A is a schematic diagram of the cell cycle after CT-26 was treated with different methods. Figure 5 B is a statistical comparison chart of cell cycle distribution. Figure 5 It can be seen that compared with RCM, the proportion of cells in the G2 / M phase decreased significantly after Pt517CS stimulation, and the proportion of cells in the G0 / G1 phase increased significantly, indicating that Pt517CS can arrest the CT-26 cell cycle in the G0 / G1 phase, preventing it from entering the S phase for DNA synthesis. Cell growth and division will be stopped, affecting the normal proliferation of tumor cells.

[0084] Example 2: The improving effect of Clostridium gracilis CGMCC No. 31232 on colorectal cancer in transplanted mice 1. Experimental design of the intervention of Clostridium tiliaceus CGMCC No.31232 on colorectal cancer xenograft mice A colorectal cancer xenograft model was established in BALB / c mice, which were then gavaged with a suspension of Clostridium gracilis CGMCC No. 31232. The experimental design was as follows: 18 5-6-week-old female SPF-grade BALB / c mice were acclimated to an SPF environment at 25°C with a 12-h light / dark cycle for 4 days and then randomly divided into three groups (n=6): a control group (NC group), a model group (PBS group), and a CGMCC No. 31232-treated group (Pt517 group). The right abdominal hair of mice in the PBS and Pt517 groups was cleaned with depilatory cream. Two days after depilation, mice in the PBS and Pt517 groups were subcutaneously injected with 0.2 mL of CT-26 colorectal cancer cells (5×10 5The transplanted tumor animal model was established. The mice were then gavaged for 15 consecutive days (1-15 days) at 10:00 am every day, with 0.2 mL per mouse. The NC and PBS groups were gavaged with PBS solution, and the Pt517 group was gavaged with CGMCC No.31232 bacterial suspension (approximately 1×10 8 CFU / mouse) were gavaged. The long diameter a (mm) and short diameter b (mm) of the tumor were measured with a vernier caliper every 2 days, and the tumor volume was calculated: tumor volume (mm 3 )=1 / 2×a×b 2 .

[0085] When the tumor volume reaches 2000 mm 3 At approximately 14 days, mice were sacrificed in accordance with animal ethics guidelines. Blood samples were collected via ocular bleeding and allowed to stand at room temperature for 1–3 hours to allow for coagulation and stratification. Serum was then separated by centrifugation at 3000 rpm for 15 minutes at 4°C. The supernatant was transferred to a clean centrifuge tube and sent to Beijing Biomarker Biotechnology Co., Ltd. for liquid chromatography-mass spectrometry and targeted mass spectrometry analysis for subsequent serum metabolomics analysis. Subcutaneous tumors were excised and weighed. A portion of the tumor was placed in a sterile centrifuge tube, passed through liquid nitrogen, and stored at −80°C for subsequent cytokine analysis. A portion of the tumor was immersed in 4% paraformaldehyde for subsequent histopathological analysis. The mouse peritoneum was opened, and the cecum was isolated. The cecal contents were collected in a sterile centrifuge tube and frozen at −80°C for subsequent intestinal microbiome analysis.

[0086] 2. Effect of Clostridium gracilis CGMCC No. 31232 on tumor formation in mice bearing colorectal cancer xenografts After oral administration of Clostridium tiliaceum CGMCC No.31232, the mice showed good daily activity and mental state, and the intervention of Clostridium tiliaceum CGMCC No.31232 had no obvious toxic side effects on the mice. Figure 6 The results of the effect of Clostridium gracilis on tumor formation in mice with colorectal cancer transplants are shown. Figure 6 A and Table 1, D 14 -D 16 On the second day, the tumor volume of the Pt517 group was significantly lower than that of the PBS group (P<0.05). Figure 6 Figure B is a comparison of the tumor sizes of mice in the PBS group and the Pt517 group. It can be seen that after 15 days of intervention with Clostridium gracilis, the tumor size of mice in the Pt517 group was significantly smaller than that in the PBS group. Figure 6 C is D 16A comparison of the tumor weights of mice in the PBS group and the Pt517 group showed that the tumor weight of the Pt517 group was significantly lower than that of the PBS group (0.75±0.18 vs 1.24±0.07, P<0.05).

[0087] Table 1 Statistics of mouse tumor volume (mm 3 )

[0088] Note: Compared with the PBS group, *, P < 0.05; **, P < 0.01.

[0089] 3. Effects of Clostridium gracilis CGMCC No. 31232 on tumor tissue pathology and Ki-67 staining results in mice bearing colorectal cancer transplants Mouse tumor tissues were removed from a 4% paraformaldehyde fixative, dehydrated, paraffin-infused, embedded, and sectioned. Hematoxylin-eosin (HE) staining and Ki-67 immunohistochemical staining were performed. The pathological morphology of the tumor tissues was observed under an optical microscope at 200x and 400x magnifications, and tumor slice images were acquired using a panoramic slice scanner. Figure 7 The following are HE staining images, Ki-67 protein immunohistochemical staining images and Ki-67 positive cell H-Score grading images of the tumors of the two groups of mice. Figure 7 Figure A is a pathological image of mice treated with PBS (200×): The image shows that the nuclei of tumor cells in the tumor tissue are irregular in shape, large in size, with a high nuclear-cytoplasmic ratio, and a large number of nuclear division figures; small focal hemorrhages can be seen locally. Figure 7 B is a pathological image of mice treated with Clostridium gracilis (200×); the image shows that a small number of tumor cells in the tumor tissue have irregular nuclei, large size, and high nuclear-cytoplasmic ratio; a small number of cells with small focal necrosis can be seen locally, with condensed, fragmented, and dissolved nuclei, increased eosinophilia, and unclear structure. Figure 7 Pathological HE staining in A and B showed that the tumor tissues of the mice in the Pt517 group had larger necrotic areas and smaller hemorrhagic areas compared with those in the PBS group.

[0090] The expression of Ki-67 is closely related to aggressive tumor biology and tumor proliferation, and is usually assessed by immunohistochemical staining in studies. Figure 7 Figure C is an immunohistochemical staining image of Ki-67 protein in mice treated with PBS (400×): The image shows that the tumor cells are closely arranged, the tumor cell density is high, and the dark Ki-67 positive cells account for a large proportion. Figure 7D is an immunohistochemical staining image of Ki-67 protein in mice treated with Clostridium gracilis (400×). The image shows loosely arranged tumor cells with a low density, and a relatively small proportion of darker Ki-67-positive cells. Based on the intensity of nuclear staining, tumor cells can be divided into Ki-67-negative and Ki-67-positive cells. The Ki-67-positive cell rate reflects the expression level of Ki-67 in the tissue and, therefore, the proliferation capacity of tumor cells. Figure 7 Figure E compares the Ki-67-positive cell ratios in the PBS and Pt517 groups. The Ki-67-positive cell ratio in the PBS group was 18.72±1.06%, and in the Pt517 group was 14.04±0.43%. Treatment with Paraclostridium tiliaceum CGMCC No. 31232 significantly reduced the Ki-67-positive cell ratio in tumor tissue (P<0.05), indicating that treatment with Paraclostridium tiliaceum CGMCC No. 31232 reduced the proliferation of tumor cells in mice bearing colorectal cancer xenografts.

[0091] 4. Effect of Clostridium gracilis CGMCC No.31232 on cytokine levels in tumor tissues of mice bearing colorectal cancer transplants Tumor immune factors play a crucial role in tumor development, progression, and immune escape. These factors not only influence tumor cell growth and proliferation but also regulate immune cell activity within the tumor microenvironment, thereby influencing antitumor immune responses. To investigate the effects of Clostridium gracilis CGMCC No. 31232 on immune factors in mice bearing colorectal cancer xenografts, we determined the levels of these factors in tumor homogenates using an ELISA assay. The specific experimental method was as follows: 0.1 g of tumor tissue was collected from each mouse, rinsed with pre-chilled PBS, minced, and ground on ice to form a homogenate for cytokine quantification. The homogenate was centrifuged at 5000 rpm for 5–10 minutes at 4°C. The supernatant was then collected and the concentrations of various cytokines in the tumor homogenate were determined using ELISA kits according to the manufacturer's instructions.

[0092] Figure 8 A comparison of cytokine levels in mouse tumor tissue after PBS and Paraclostridium tiliaceum intervention is shown. Anti-tumor immune factors such as TNF-α, IFN-γ, and CD8 were significantly higher in the Pt517 group than in the PBS group, while immunosuppressive immune factors such as IL-6, IL-10, and TGF-β were significantly lower in the PBS group. These results indicate that Paraclostridium tiliaceum CGMCC No. 31232 alters immune factors in tumors of mice bearing colorectal cancer xenografts.

[0093] 5. Effects of Clostridium gracilis CGMCC No. 31232 on the intestinal flora of mice bearing colorectal cancer transplants The intestinal microbiome plays a crucial role in the development, progression, and treatment of colorectal cancer. To investigate the effects of Clostridium gracilis CGMCC No. 31232 on the intestinal microbiome of mice bearing colorectal cancer xenografts, 16S rRNA gene sequencing was performed on the cecal contents of these mice. Bacterial DNA was extracted from the cecal contents of the mice using a fecal genomic DNA extraction kit according to the kit's instructions. The extracted DNA was then sent to Beijing Biomark Biotechnology Co., Ltd. for sequencing.

[0094] The Chao1 index and Shannon index were used to measure the α diversity of the mouse cecal contents. Figure 9 As shown in A and B, compared with the NC group, the Chao1 index and Shannon index of the PBS group showed no significant changes (P>0.05). At the same time, there was no statistically significant difference in the Chao1 index and Shannon index between the Pt517 group and the PBS group (P>0.05), indicating that the intervention of Clostridium tenuis CGMCC No. 31232 had no effect on the species richness and uniformity of the intestinal flora in mice with colorectal cancer transplanted tumors.

[0095] PCoA was used to analyze the β diversity of intestinal flora in mice with colorectal cancer transplants. Figure 9 As shown in Figure C. The distribution distance between samples in the PBS and NC groups was greater, indicating that colorectal cancer cell inoculation altered the intestinal microbiome structure of mice. Compared to the PBS group, the distribution distance between samples in the Pt517 and NC groups was closer, suggesting that intervention with Clostridium truncatum CGMCC No. 31232 can alter the intestinal microbiome structure of mice bearing colorectal cancer xenografts and normalize it.

[0096] Figure 9 Figure D shows the top 10 relative abundances of the intestinal microbiota in mice with colorectal cancer transplants. The most abundant genus in the NC group was Alternaria ( Alistipes , 30.39%), followed by the Lachnospiraceae NK4A136 group ( Lachnospiraceae _NK4A136_group, 10.33%), Bacteroides spp. Bacteroides , 7.57%), uncultured rumen bacteria (uncultured_rumen_bacterium, 6.77%), and Lactobacillus spp. ( Lactobacillus , 5.66%). The most abundant genus in the PBS group was Lactobacillus ( Lactobacillus , 21.57%), followed by Lactobacillus sp. ( Ligilactobacillus , 18.61%), another genus ( Alistipes, 12.43%), Bacteroides ( Bacteroides ,9.94%) and uncultured Bacteroidetes Bacteroidales _bacterium, 7.94%). The most abundant genus in the Pt517 group was Lactobacillus ( Ligilactobacillus , 20.11%), and another genus Mycobacterium ( Alistipes , 18.79%), uncultured rumen bacteria (uncultured_rumen_bacterium, 15.29%), Lachnospiraceae NK4A136 group ( Lachnospiraceae _NK4A136_group, 8.36%) and Lachnospiraceae UCG_006 group ( Lachnospiraceae _UCG_006, 5.50%). Compared with the NC group, the PBS group had a different genus ( Alistipes ) were significantly decreased (P<0.05); the relative abundance of other genus ( Alistipes The relative abundance of uncultured rumen bacteria ( ) increased compared with the PBS group, but the difference was not statistically significant (P>0.05). The relative abundance of uncultured rumen bacteria ( uncultured_rumen_bacterium ) decreased in the PBS group compared with the NC group, but the difference was not statistically significant. The relative abundance of uncultured rumen bacteria ( uncultured_rumen_bacterium ) in the Pt517 group was significantly higher than that in the PBS group (P<0.05). These results indicate that treatment with Paraclostridium tenuis CGMCC No. 31232 altered the genus-level structure of the mouse intestinal microbiota.

[0097] LEfSe analysis was used to screen the taxonomic units with statistically significant differences in the intestinal flora of each group of mice at the species level (LDA>3, P<0.05). Figure 9 As shown in E, at the species level, Odessa visceralis ( Odoribacter splanchnicus ) and another Cladosporium genus CHKCI003 ( Alistipes sp CHKCI003) were significantly increased in the NC group, while the relative abundance of bacteria AC2043 ( Christensenellaceae R 7 group bacteriumAC2043) and Bacteroides pasadena ( Paenibacillus pasadenensis ) increased significantly, and the relative abundance of Lachnospiraceae bacteria G11 ( Lachnospiraceae bacterium G11), Clostridium ( Clostridium fusiformis ), Adkretzschie's bacterium ( Adlercreutzia muris )、Cecal Enterobacter B7( Enterorhabdus caecimuris B7), Colidectes ASF500 ( Colidextribacterbacterium ASF500), Clostridium sp ASF356 and Parabacteroides guineensis Parabacteroides goldsteinii ) were significantly enriched in the Pt517 group. Oral administration of Clostridium gracilis Pt517 can increase the relative abundance of some bacteria with probiotic properties, such as the Parabacteroides archaeopteris mentioned above ( Parabacteroides goldsteinii , P<0.05), Lachnospiraceae G11 ( Lachnospiraceae bacterium G11, P<0.01) and cecal Enterobacterium B7 ( Enterorhabdus caecimuris B7, P < 0.05) ( Figure 9 The intervention of Paraclostridium gracilis CGMCC No.31232 can also significantly reduce the relative abundance of bacteria with pathogenic potential, such as the Christensenaceae R7 group bacteria AC2043 ( Christensenellaceae R 7 group bacterium AC2043, P<0.05) ( Figure 9 These results indicate that Clostridium gracilis CGMCC No. 31232 can regulate the intestinal flora of mice, increase the relative abundance of beneficial bacteria, reduce the relative abundance of potential pathogenic bacteria, and make the intestinal flora composition of colorectal cancer transplanted mice closer to that of normal mice.

[0098] 6. Clostridium gracilis CGMCC No.31232 can produce metabolites with anti-colorectal cancer effects Many studies have shown that probiotics can secrete metabolites with anti-tumor effects and inhibit colorectal cancer. Therefore, metabolomics analysis was performed on the serum of colorectal cancer transplanted mice and Pt517CS.

[0099] To investigate the differential metabolites among the NC, PBS, and Pt517 groups, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed. Figure 10 As shown in Figure A, the OPLS-DA model score plot shows the differences in serum metabolites between the NC and PBS groups. Similarly, Figure 10 The OPLS-DA model score graph in C shows the differences in serum metabolites between the PBS group and the Pt517 group. To further investigate the changes in serum metabolites after tumor cell inoculation and intervention with Clostridium subtilis CGMCC No. 31232, differential analysis of metabolites in different groups was performed, with P < 0.05 as the screening criterion for differential metabolites. A volcano plot was used for visualization. The results are shown in Figure 3. Figure 10Next, the KEGG database was used to find the metabolic pathways involved in the differential metabolites between the Pt517 group and the PBS group. The top 10 significantly enriched metabolic pathways were selected according to the enrichment factor (Rich Factor) to visualize the data ( Figure 10 E). Figure 10 As shown in Figure E, differential metabolites between the Pt517 and PBS groups were significantly enriched in metabolic pathways such as the biosynthesis of unsaturated fatty acids, linoleic acid metabolism, central carbon metabolism, and choline metabolism. Among them, the metabolic pathway with the most enriched and most significant metabolites was the unsaturated fatty acid biosynthesis pathway, which may be the key to the anti-cancer effect of Clostridium gracilis CGMCC No. 31232 in the colorectal cancer xenograft mouse model.

[0100] Therefore, the differential metabolites in the unsaturated fatty acid biosynthesis pathway were hierarchically clustered, and a heat map was made to more intuitively display the expression levels of the differential metabolites in the Pt517 group and the PBS group ( Figure 10 F). It is worth noting that compared with the PBS group, the levels of palmitic acid and stearic acid in the Pt517 group were significantly increased (P<0.05). The levels of palmitic acid and stearic acid in the Pt517CS group were also significantly increased compared with the RCM control group (P<0.05, Table 2), indicating that Paraclostridium gracilis CGMCC No. 31232 can produce palmitic acid and stearic acid in vivo and in vitro. Through genomic analysis of Paraclostridium gracilis CGMCC No. 31232, it was found that Paraclostridium gracilis CGMCC No. 31232 has the key enzymes for producing palmitic acid and stearic acid, further proving that Paraclostridium gracilis CGMCC No. 31232 has the ability to produce these two unsaturated fatty acids ( Figure 10 G).

[0101] Table 2 Contents of long-chain fatty acids identified in Pt517CS and RCM (ng / mL)

[0102] Literature indicates that palmitic acid and stearic acid have anti-cancer effects both in vivo and in vitro, suggesting that they may contribute to the anti-CRC effect of Clostridium gracilis CGMCC No. 31232. Therefore, we investigated the effects of palmitic acid and stearic acid on the proliferation of colorectal cancer cells in vitro. The results showed that compared with the ethanol solvent, 100 μM palmitic acid or stearic acid significantly reduced the proliferation activity of CT26 cells (P < 0.001, Figure 10 H), and the inhibitory effects of palmitic acid and stearic acid on the proliferation of CT26 cells were concentration-dependent ( Figure 10 In conclusion, C. tenuissima CGMCC No. 31232 can produce metabolites (palmitic acid and stearic acid) with anti-CRC effects and change the serum composition of mice bearing colorectal cancer xenografts.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Clostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in preparing a product for improving or treating colorectal cancer; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

2. Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for preventing colorectal cancer; the paraclostridium slenderum ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

3. Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for inhibiting proliferation, cloning and / or migration of colorectal cancer cells; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

4. Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for promoting apoptosis of colorectal cancer cells; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

5. Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for blocking the colorectal cancer cell cycle; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

6. Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for regulating the secretion of immune factors in patients with colorectal cancer; the Paraclostridium tenuis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

7. Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of a product for regulating the intestinal flora of patients with colorectal cancer; the Paraclostridium tenuis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

8. Paraclostridium gracilis ( Paraclostridium tenue ) Application of Pt517 in the preparation of products for increasing the content of palmitic acid and / or stearic acid; the Paraclostridium gracilis ( Paraclostridium tenue )The deposit number of Pt517 is CGMCC No.31232.

9. The product according to any one of claims 1 to 8, characterized in that The product is a drug.

10. The product according to any one of claims 1 to 8, characterized in that: The active ingredients of the product include the paraclostridium gracilis ( Paraclostridium tenue ) Pt517 or a culture thereof.

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