Application of intestinal flora metabolite glutaric acid in prevention and treatment of colorectal cancer

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of intestinal flora metabolite glutaric acid in preventing and treating colorectal cancer. The glutaric acid can obviously inhibit the proliferation of colorectal cancer cells, is in concentration dependence and time dependence, and reduces the proportion of EdU-positive red stained cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of glutaric acid, a metabolite of intestinal flora, in the prevention and treatment of colorectal cancer. Background Art

[0002] Colorectal cancer is a common malignant tumor in the gastrointestinal tract. Among them, more than 80% of patients are already in the middle and late stages at the time of diagnosis, and more than 40% of patients have liver and lung metastasis when discovered. For colorectal cancer, in addition to surgical resection, the current mainstream treatment methods include widely used chemotherapy and immunotherapy, but these treatment options still have obvious disadvantages. Surgical treatment must remove a large amount of normal tissue or even the entire organ, and there is still a risk of spread and metastasis. Chemotherapy and radiotherapy can seriously damage the function of tissues and organs such as the immune system. Immunotherapy is only suitable for a limited number of people, and immune-related adverse reactions also need to be paid attention to and managed. Therefore, it is still necessary to explore appropriate treatment methods for colorectal cancer.

[0003] The intestinal flora is considered to be one of the key factors regulating host health, and the disorder of intestinal flora is associated with a variety of diseases. Studies have shown that specific intestinal bacteria are strongly associated with obesity, type 2 diabetes, Crohn's disease, neurodegenerative diseases and even cancer. The intestinal bacterium Akkermansia muciniphila is a Gram-negative, obligate anaerobic intestinal bacterium that was isolated and identified in 2004. It is mainly found in the intestines of humans and other mammals, and uses mucin in intestinal mucus as its main energy source. Studies have reported that Akkermansia muciniphila can inhibit tumor progression by reducing DNA damage and promoting tumor cell apoptosis.

[0004] The acetyltransferase Amuc_2172 secreted by Akkermansia muciniphila can induce the secretion of heat shock protein 70, promote the activity of CD8+ cytotoxic T lymphocytes, reshape the tumor microenvironment, and inhibit the occurrence of colorectal cancer. Butyrate, a metabolite of Akkermansia muciniphila, reduces the expression of miR-183, increases the expression of heat shock protein B4, promotes cell apoptosis, and ultimately inhibits the malignant progression of colorectal cancer through the miR-183 / DNAJB4 axis. The metabolites produced by Akkermansia muciniphila in the course of its life activities play an important role in the occurrence and development of colorectal cancer. The present invention proposes glutaric acid, a metabolite of Akkermansia muciniphila that can inhibit the progression of colorectal cancer, which is expected to provide more options for the treatment of colorectal cancer. Summary of the invention

[0005] The object of the present invention is to provide the use of glutaric acid in preparing a medicine for preventing and / or treating colorectal cancer.

[0006] The results of the study showed that glutaric acid, a metabolite of the intestinal bacteria Akkermansia muciniphila, has a significant inhibitory effect on colorectal cancer.

[0007] The pharmaceutical application of the present invention comprises:

[0008] Glutaric acid can significantly inhibit the proliferation of colorectal cancer cells in a concentration-dependent and time-dependent manner, and reduce the proportion of EdU-positive red-stained cells.

[0009] Glutaric acid inhibits the migration rate and number of migrating cells of colorectal cancer cells.

[0010] Glutaric acid inhibits the volume and weight of mouse tumors.

[0011] Glutaric acid reduces tumor tissue necrosis and the number of pathological mitotic cells.

[0012] Glutaric acid reduces the expression of Ki67 in mouse tumor tissues.

[0013] The efficacy of the drug of the present invention is obtained after a large number of experimental screenings:

[0014] 1. Screening of intestinal bacterial metabolites with anti-cancer effects

[0015] The metabolites of the intestinal bacteria Akkermansia muciniphila were intersected with the metabolites of the intestinal flora of colorectal cancer patients in the Human Metabolome Database (HMDB) to obtain 5 common metabolites, and the top 5 metabolites were selected according to their content: xanthine nucleoside, uric acid, thymidine-5'-monophosphate, citraconic acid, and glutaric acid. Metabolites with anti-cancer effects were preliminarily screened through cell experiments.

[0016] Human colorectal cancer cell line HCT116 and mouse colorectal cancer cell line MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture). After the cells grew to the logarithmic phase, they were digested with trypsin to form a single cell suspension and inoculated into 96-well plates (6×10 3 ), after culturing in an incubator (37°C, 95% humidity, 5% CO2) for 24 hours, different concentrations of intestinal bacteria Akkermansia muciniphila metabolites were added and cultured for another 24 hours. The cell activity was detected using the CCK8 cell viability kit, and the survival rate and IC50 value of colorectal cancer cells under the action of different metabolites were calculated. Cell survival rate = (OD value of the experimental well - OD value of the blank well) / (OD value of the control well - OD value of the blank well) × 100%.

[0017] At the same time, human normal intestinal epithelial cells NCM460 were used to screen metabolites with anti-cancer effects and low toxicity. Human normal intestinal epithelial cells NCM460 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture). After the cells grew to the logarithmic phase, they were digested with trypsin to form a single cell suspension and inoculated into a 96-well plate (8×10 3 ) in an incubator (37°C, 95% humidity, 5% CO2) for 24 h, and then different concentrations of intestinal bacteria Akkermansia muciniphila metabolites were added for 24 h. The cell activity was detected using the CCK8 cell viability kit, and the cell survival rate and IC50 value were calculated.

[0018] 2. In vitro verification of intestinal flora metabolite glutaric acid inhibiting colorectal cancer

[0019] (1) Using CCK8 cell viability kit to detect the time dependence of glutaric acid inhibition of colorectal cancer cells

[0020] Human colorectal cancer cell line HCT116 and mouse colorectal cancer cell line MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture). After the cells grew to the logarithmic phase, they were digested with trypsin to form a single cell suspension and inoculated into 96-well plates (6×10 3 ) and cultured in an incubator (37°C, 95% humidity, 5% CO2) for 24 h. Then, glutaric acid (6.5 mmol / L) was added for 24 h, 48 h, and 72 h, respectively. HEPES was used to adjust the pH of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. The CCK8 cell viability kit was used to detect cell activity and calculate the cell survival rate.

[0021] (2) EdU Imaging Kit was used to detect the effect of glutaric acid on the proliferation of colorectal cancer cells

[0022] The colorectal cancer cell lines HCT116 and MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture) until the logarithmic growth phase, and then digested with trypsin to form a single cell suspension and inoculated into a 6-well plate (1×10 4), incubated in an incubator (37°C, 95% humidity, 5% CO2) for 24 hours, glutaric acid was added, and HEPES was used to adjust the pH of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. After incubation for 48 hours, the culture medium was changed to 50μmmol / L EdU staining solution to mark the DNA of proliferating cells, and incubated at 37°C for 2 hours. Fixed with 4% paraformaldehyde for 30 minutes, permeated with 0.3% Triton-X, added the reaction solution and incubated for 30 minutes, added Hoechst33342 to stain the nucleus for 10 minutes, and observed and collected images under a fluorescence microscope.

[0023] (3) Cell scratch assay to detect the effect of glutaric acid on the migration of colorectal cancer cells

[0024] After culturing the colorectal cancer cell lines HCT116 and MC38 to the logarithmic growth phase using RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture), trypsin was used to digest the cells into a single cell suspension and inoculated into a 24-well plate and cultured in an incubator (37°C, 95% humidity, 5% CO2). When the cell density is 75%, scratch the cell layer vertically with a 10μL gun tip, rinse gently with PBS, add glutaric acid to treat the cells, and use HEPES to adjust the pH of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. Observe and take pictures under a microscope to record the changes in the scratch at 0h, 24h and 48h, and calculate the migration rate of the cells at 24h and 48h. Migration rate = (0h scratch width-scratch width at the observation time point) / 0h scratch width.

[0025] (4) Transwell assay to detect the effect of glutaric acid on the migration of colorectal cancer cells

[0026] Colorectal cancer cell lines HCT116 and MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture) until the logarithmic growth phase, then digested with trypsin to form a single cell suspension and inoculated into the chamber of a cell culture plate (HCT116: 2×10 5 , MC38: 1.5×10 5 ), add medium containing glutaric acid, and use HEPES to adjust the pH value of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. Add medium containing 20% ​​serum to the lower chamber. Culture in a cell culture incubator (37°C, 5% CO2) for 24 hours. After fixing the cells with paraformaldehyde, stain with crystal violet, and gently wipe the unpenetrated cells in the upper chamber with a cotton swab, observe and collect images under a microscope, and use Image J software to calculate the number of migrated cells.

[0027] (5) One-step TUNEL FITC Apoptosis Detection Kit was used to detect the effect of glutaric acid on apoptosis of colorectal cancer cells

[0028] Colorectal cancer cells were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture) until the logarithmic growth phase, then digested with trypsin to form a single cell suspension and inoculated into cell culture plates (1×10 4 ), cultured in an incubator (37°C, 95% humidity, 5% CO2) for 24 h, and then treated with glutaric acid for 48 h. HEPES was used to adjust the pH of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. Cells were fixed with 4% paraformaldehyde at 4°C for 25 min, permeabilized with 0.2% Triton X-100 for 5 min, and then treated with 1×

[0029] The cells were equilibrated with Equilibration Buffer at room temperature for 20 min, and 200 μL of labeling reaction solution was added and incubated at 37°C in the dark for 60 min. The cell nuclei were counterstained with 2 μg / mL DAPI at room temperature for 5 min, and the cells were washed with PBS three times, each time for 5 min. The distribution of green fluorescence was observed under a fluorescence microscope and images were collected.

[0030] 3. In vivo verification of intestinal bacterial metabolite glutaric acid inhibiting colorectal cancer

[0031] (1) Establishing a transplanted tumor model to detect the effect of glutaric acid on mouse tumors

[0032] MC38 cells in the logarithmic growth phase were obtained and digested with trypsin to form a single cell suspension, which was then subcutaneously injected into the upper right thigh of C57BL / 6 mice (8×10 5 ), and when the tumor volume of the mice increased to 100 mm 3 Glutaric acid (100 mg / kg) was injected intraperitoneally every two days, while the control group was injected with an equal volume of saline. The model construction process is as follows Figure 7 As shown in A. During the feeding process, the weight of mice was weighed and recorded weekly. When the tumor volume of mice in the control group increased to 2000mm 3 The tumors of mice in each group were removed and weighed, and the long and short diameters of the tumors were measured with a vernier caliper to calculate the tumor volume. Tumor volume = (long diameter × short diameter) 2 ) / 2.

[0033] (2) HE staining was used to detect the effect of glutaric acid on mouse tumor tissue

[0034] HE staining was used to detect the effect of glutaric acid on mouse tumor tissue. After dehydration, embedding and sectioning of the tumor tissue, the following steps were performed:

[0035] 1) Dewaxing and hydration: First, bake the slides in a 60°C oven for 30 min, soak them in xylene I and xylene II for 15 min, soak them in 100% ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 min, and rinse them with distilled water three times.

[0036] 2) Staining: Stain with hematoxylin for 2 min, rinse with running water for 5 min, dry and then stain with eosin for 10 s, rinse with running water for 5 min.

[0037] 3) Dehydration: Soak in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 2 minutes, and then soak in xylene I and xylene II for 10 minutes.

[0038] 4) Sealing: Use neutral gum to seal the slides, bake at 37°C overnight and then store at room temperature.

[0039] 5) Taking photos: Observe and collect images under a microscope.

[0040] (3) Immunohistochemistry was used to detect the expression of Ki67 in tumor tissues

[0041] Immunohistochemistry kit was used to detect the expression of Ki67 in tumor tissues to verify the effect of glutaric acid on tumor cell proliferation in vivo. After dehydration, embedding and sectioning of tumor tissues, the following steps were performed:

[0042] 1) Dewaxing and hydration: First, take out the slices, make a record on the slide and insert them into the slide rack, bake them in a 60°C oven for 30 min, soak them in xylene I and xylene II for 15 min, soak them in 100% ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 min, and rinse them with distilled water three times.

[0043] 2) Antigen repair: Place the tissue sections in 200 mL EDTA antigen repair solution and preheat at low temperature for 1 min, high temperature for 2 min, and low temperature for 10 min. Take out the tissue sections and cool them naturally to room temperature. Place the sections in PBS and rinse them three times.

[0044] 3) Removal of endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocker to the sliced ​​tissue, incubate at room temperature for 30 minutes, and rinse three times with PBS, each time for 5 minutes.

[0045] 4) Serum blocking: After the slices are slightly dry, add an appropriate amount of normal goat serum working solution, block at 37°C for 20 minutes, and wash with PBS three times, each time for 3 minutes.

[0046] 5) Incubate with antibodies: Aspirate the surrounding liquid, add an appropriate amount of primary antibody diluent (1:500) to soak the slices, store in a 4°C refrigerator overnight, take out the slices and rewarm for 1 hour, rinse with PBS three times, 5 minutes each time. Incubate with biotin-labeled goat anti-rabbit IgG at 37°C for 20 minutes, rinse with PBS three times, 5 minutes each time. Aspirate the surrounding liquid, add an appropriate amount of horseradish enzyme-labeled streptavidin and incubate for 20 minutes, rinse with PBS three times, 5 minutes each time.

[0047] 6) DAB staining: wipe off the liquid on the slice, add appropriate amount of DAB staining solution, let it stand for staining for 3 minutes, observe under a microscope to avoid over-staining, and wash with tap water for 5 minutes.

[0048] 7) Hematoxylin counterstaining: Hematoxylin counterstaining for 2 minutes, and then washing with distilled water for 3 minutes.

[0049] 8) Dehydration: Soak the tissue sections in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 2 minutes, and then soak them in xylene I and xylene II for 10 minutes.

[0050] 9) Sealing: After the slices are dried, add neutral resin, bake at 37°C overnight, and store at room temperature.

[0051] 10) Taking photos: Observe and collect images under a microscope.

[0052] The present invention proposes that the metabolite glutaric acid of intestinal bacteria Akkermansia muciniphila has an effect on colorectal cancer

[0053] It has a significant inhibitory effect. Glutaric acid can significantly inhibit the proliferation of colorectal cancer cells in a concentration-dependent and time-dependent manner, reduce the proportion of EdU-positive red-stained cells, inhibit the migration rate of colorectal cancer cells and the number of migrating cells, inhibit the volume and weight of mouse transplanted tumors, reduce tumor tissue necrosis and the number of pathological nuclear division cells, and reduce the expression of Ki67 in mouse tumor tissues.

[0054] Explanation of some English abbreviations or terms in the manual:

[0055] Akkermansia muciniphila: Akkermansia muciniphila.

[0056] HCT116: A human colorectal cancer cell line isolated by M. Brattain et al. from an adult male patient with colorectal cancer. The cell morphology is epithelial-like and grows adherently.

[0057] MC38: mouse colorectal cancer cell line.

[0058] NCM460: Normal human intestinal epithelial cells, derived from the normal colon mucosa of a 68-year-old Hispanic male. CellCounting Kit-8: This kit contains the water-soluble tetrazolium salt WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolium monosodium salt], which can be used for simple and accurate cell proliferation and toxicity analysis.

[0059] RPMI1640: cell culture medium.

[0060] HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, is an amphoteric organic chemical buffer that can be used as a buffering reagent.

[0061] EdU Imaging Kit: The thymidine nucleoside analog EdU (5-ethynyl-2'-deoxyuridine) is not affected by harsh DNA denaturation conditions. EdU can be incorporated into the DNA chain during DNA synthesis. It uses HyperFluor TM 488azide is linked to EdU to fluorescently label the DNA of proliferating cells, which can be observed under a fluorescence microscope.

[0062] Hoechst33342: a live cell nuclear marker dye.

[0063] Triton X-100: Triton X-100, a non-ionic surfactant, is used to permeabilize cell membranes.

[0064] Transwell assay: Transwell migration or invasion assay is used to study the migration, chemotaxis and invasion of cells in response to various stimuli such as growth factors, chemokines or extracellular matrix components.

[0065] One-step TUNEL FITC Apoptosis Detection Kit: One-step TUNEL cell apoptosis detection kit, detect the apoptosis of cultured adherent cells or suspension cells.

[0066] DAPI: 4',6-diamidino-2-phenylindole, is a blue fluorescent DNA dye.

[0067] PBS: phosphate buffered saline.

[0068] HE staining: Hematoxylin-eosin staining method. Hematoxylin staining solution is alkaline, which makes the chromatin in the cell nucleus and the nucleic acid in the cytoplasm purple-blue; eosin is an acidic dye, which makes the components in the cytoplasm and extracellular matrix red. IHC Kit: Immunohistochemistry Kit.

[0069] Ki67: Ki67 antibody is a cell marker for proliferation. Immunohistochemistry is used to evaluate nuclear expression of Ki67 to assess tumor proliferation.

[0070] EDTA: Ethylenediaminetetraacetic acid, used for antigen retrieval.

[0071] DAB: 3,3N-Diaminobenzidine Tertrahydrochloride, is a common substrate for horseradish peroxidase. Under the catalysis of horseradish peroxidase, DAB will produce a brown precipitate. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 .Effects of the metabolites of intestinal bacteria Akkermansia muciniphila on the proliferation of colorectal cancer cellsA. Glutaric acid inhibits the activity of human colorectal cancer cells HCT116B. Glutaric acid inhibits the activity of mouse colorectal cancer cells MC38C. Low concentrations of glutaric acid have no significant effect on the activity of normal human intestinal epithelial cells NCM460D. Citraconic acid inhibits the activity of human colorectal cancer cells HCT116E. Citraconic acid inhibits the activity of mouse colorectal cancer cells MC38F. Xanthine nucleoside promotes the activity of human colorectal cancer cells HCT116G. Xanthine nucleoside promotes the activity of mouse colorectal cancer cells MC38H. Thymidine-5'-monophosphate promotes the activity of human colorectal cancer cells HCT116I. Thymidine-5'-monophosphate promotes the activity of mouse colorectal cancer cells MC38J. Uric acid has no significant effect on the activity of human colorectal cancer cells HCT116K. Uric acid has no significant effect on the activity of mouse colorectal cancer cells MC38

[0073] Figure 2 .Glutaric acid has a time-dependent effect on the proliferation of colorectal cancer cells. A. Activity of HCT116 cells B. Activity of MC38 cells *P<0.033, **P<0.002, ***P<0.001

[0074] Figure 3 .EdU Imaging Kit to detect the effect of glutaric acid on the proliferation of colorectal cancer cells A. EdU-positive HCT116 red-stained cells B. EdU-positive MC38 red-stained cells Contrl, control group; Glutaric Acid, glutaric acid treatment group. **P<0.002, ***P<0.001

[0075] Figure 4 . Cell scratch assay to detect the effect of glutaric acid on the migration of colorectal cancer cells A. Migration of HCT116 cells B. Migration of MC38 cells Contrl, control group; Glutaric Acid, glutaric acid treatment group. ***P<0.001

[0076] Figure 5 .Transwell assay to detect the effect of glutaric acid on the migration of colorectal cancer cells A. Migration of HCT116 cells B. Migration of MC38 cells Contrl, control group; Glutaric Acid, glutaric acid treatment group. ***P<0.001 Figure 6 TUNEL apoptosis kit was used to detect the effect of glutaric acid on apoptosis of colorectal cancer cells. A. Apoptosis of HCT116 cells B. Apoptosis of MC38 cells Contrl, control group; Glutaric Acid, glutaric acid treatment group.

[0077] Figure 7 Construction of transplant tumor model to detect the effect of glutaric acid on mouse tumorsA. Construction of transplant tumor model and drug administration processB. Exfoliated transplant tumorC. Changes in transplant tumor volumeD. Weight of transplant tumorE. Changes in mouse body weightCRC: colorectal cancer group; Glutaric Acid, glutaric acid treatment group.

[0078] Figure 8 .HE staining to detect the effect of glutaric acid on mouse tumor tissue CRC, colorectal cancer group; Glutaric Acid, glutaric acid treatment group.

[0079] Fig. 9 .Immunohistochemistry detection of the effect of glutaric acid on mouse tumor proliferationCRC, colorectal cancer group; Glutaric Acid, glutaric acid treatment group. DETAILED DESCRIPTION

[0080] The present invention is further illustrated by the following examples.

[0081] The product and preparation method of the present invention are further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0082] Example 1 Screening of intestinal bacterial metabolites with anticancer effects

[0083] The metabolites of the intestinal bacteria Akkermansia muciniphila were intersected with the metabolites of the intestinal flora of colorectal cancer patients in the Human Metabolome Database (HMDB), and the top five metabolites were selected according to their content: xanthine nucleoside, uric acid, thymidine-5'-monophosphate, citraconic acid, and glutaric acid. Metabolites with anti-cancer effects were preliminarily screened through cell experiments.

[0084] Human colorectal cancer cell line HCT116 and mouse colorectal cancer cell line MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture). After the cells grew to the logarithmic phase, they were digested with trypsin to form a single cell suspension and inoculated into 96-well plates (6×10 3 ), after culturing in an incubator (37°C, 95% humidity, 5% CO2) for 24 hours, different concentrations of intestinal bacteria Akkermansia muciniphila metabolites were added and cultured for another 24 hours. The cell activity was detected using the CCK8 cell viability kit, and the survival rate and IC50 value of colorectal cancer cells under the action of different metabolites were calculated. Cell survival rate = (OD value of the experimental well - OD value of the blank well) / (OD value of the control well - OD value of the blank well) × 100%.

[0085] At the same time, human normal intestinal epithelial cells NCM460 were used to screen metabolites with anti-cancer effects and low toxicity. Human normal intestinal epithelial cells NCM460 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture). After the cells grew to the logarithmic phase, they were digested with trypsin to form a single cell suspension and inoculated into a 96-well plate (8×10 3 ) in an incubator (37°C, 95% humidity, 5% CO2) for 24 h, and then different concentrations of intestinal bacteria Akkermansia muciniphila metabolites were added for 24 h. The cell activity was detected using the CCK8 cell viability kit, and the cell survival rate and IC50 value were calculated.

[0086] The results showed that glutaric acid, a metabolite of intestinal bacteria Akkermansia muciniphila, could significantly inhibit the proliferation of colorectal cancer cells in a concentration-dependent manner. The IC50 value for colorectal cancer cells HCT116 was 13mmol / L ( Figure 1 A), the IC50 value of the drug on colorectal cancer cells MC38 was 13mmol / L ( Figure 1 B), the IC50 value of NCM460 acting on normal intestinal epithelial cells is 15mmol / L ( Figure 1 C), but had little effect on NCM460 at low concentrations. Therefore, the 1 / 2IC50 value was selected for subsequent experiments, and the action concentrations of HCT116 and MC38 were both 6.5mmol / L. The metabolite of intestinal bacteria Akkermansia muciniphila, citraconic acid, can significantly inhibit the proliferation of colorectal cancer cells in a concentration-dependent manner ( Figure 1 D, E). Xanthine nucleosides, metabolites of the intestinal bacteria Akkermansia muciniphila, can significantly promote the proliferation of colorectal cancer cells in a concentration-dependent manner ( Figure 1F, G). Thymidine-5'-monophosphate, a metabolite of the intestinal bacterium Akkermansia muciniphila, can significantly promote the proliferation of colorectal cancer cells in a concentration-dependent manner ( Figure 1 H, I). Uric acid, a metabolite of the intestinal bacterium Akkermansia muciniphila, failed to significantly inhibit the proliferation of colorectal cancer cells ( Figure 1 J, K). The present invention is primarily concerned with glutaric acid.

[0087] Example 2 In vitro verification of intestinal flora metabolite glutaric acid inhibiting colorectal cancer

[0088] (1) Using CCK8 cell viability kit to detect the time dependence of glutaric acid inhibition of colorectal cancer cells

[0089] Human colorectal cancer cell line HCT116 and mouse colorectal cancer cell line MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture). After the cells grew to the logarithmic phase, they were digested with trypsin to form a single cell suspension and inoculated into 96-well plates (6×10 3 ) and cultured in an incubator (37°C, 95% humidity, 5% CO2) for 24 hours. Then, glutaric acid (6.5mmol / L) was added for 24 hours, 48 ​​hours and 72 hours, respectively. At the same time, HEPES was used to adjust the pH of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. The CCK8 cell viability kit was used to detect cell activity and calculate the cell survival rate. The results showed that with the extension of the action time, the inhibitory effect of glutaric acid on HCT116 cells and MC38 cells was significantly enhanced, indicating that the inhibitory effect of glutaric acid on colorectal cancer cells was time-dependent ( Figure 2 A, B). Considering the inhibitory effect and action time, the action time of 48 h was selected for subsequent experiments.

[0090] (2) EdU Imaging Kit was used to detect the effect of glutaric acid on the proliferation of colorectal cancer cells

[0091] The colorectal cancer cell lines HCT116 and MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture) until the logarithmic growth phase, and then digested with trypsin to form a single cell suspension and inoculated into a 6-well plate (1×10 4), cultured in an incubator (37°C, 95% humidity, 5% CO2) for 24 hours, then glutaric acid was added, and HEPES was used to adjust the pH of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. After continuing to incubate for 48 hours, the culture medium was changed to contain 50μmmol / L EdU staining solution to mark the DNA of proliferating cells, and incubated at 37°C for 2 hours. Fixed with 4% paraformaldehyde for 30 minutes, permeated with 0.3% Triton-X, added the reaction solution and incubated for 30 minutes, Hoechst33342 was added to stain the nucleus for 10 minutes, and observed and collected images under a fluorescence microscope. The results showed that compared with the normal control group, the proportion of EdU-positive red-stained cells in HCT116 and MC38 cells treated with glutaric acid was significantly reduced, indicating that glutaric acid can significantly inhibit the proliferation of colorectal cancer cells ( Figure 3 A, B).

[0092] (3) The cell scratch assay was used to detect the effect of glutaric acid on the migration of colorectal cancer cells.

[0093] The colorectal cancer cell lines HCT116 and MC38 were cultured to the logarithmic growth phase using RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture), and then digested with trypsin into a single cell suspension and inoculated into a 24-well plate and cultured in an incubator (37°C, 95% humidity, 5% CO2). When the cell density reached 75%, a 10μL pipette tip was used to vertically scratch the cell layer, and after gently rinsing with PBS, glutaric acid was added to treat the cells, and HEPES was used to adjust the pH of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. The changes in the scratch at 0h, 24h and 48h were observed and photographed under a microscope, and the migration distance of the cells at 24h and 48h was calculated. Migration rate = (0h scratch width - scratch width at the observation time point) / 0h scratch width. The results showed that compared with the control group, the migration rates of HCT116 and MC38 cells were significantly reduced after 24h and 48h of glutaric acid treatment ( Figure 4 A, B), indicating that glutaric acid can significantly inhibit the migration of colorectal cancer cells.

[0094] (4) Transwell assay to detect the effect of glutaric acid on the migration of colorectal cancer cells

[0095] Colorectal cancer cell lines HCT116 and MC38 were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture) until the logarithmic growth phase, then digested with trypsin to form a single cell suspension and inoculated into the chamber of a cell culture plate (HCT116: 2×10 5 , MC38: 1.5×10 5), add culture medium containing glutaric acid, and use HEPES to adjust the pH value of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. Add culture medium containing 20% ​​serum to the lower chamber. Culture in a cell culture incubator (37°C, 5% CO2) for 24 hours. After fixing the cells with paraformaldehyde, stain them with crystal violet, and gently wipe off the unpenetrated cells in the upper chamber with a cotton swab. Observe and collect images under a microscope, and use Image J software to calculate the number of migrated cells. The results showed that compared with the control group, the number of migrated HCT116 and MC38 cells treated with glutaric acid was significantly reduced ( Figure 5 ), further indicating that glutaric acid can significantly inhibit the migration of colorectal cancer cells.

[0096] (5) One-step TUNEL FITC Apoptosis Detection Kit was used to detect the effect of glutaric acid on apoptosis of colorectal cancer cells

[0097] Colorectal cancer cells were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture) until the logarithmic growth phase, then digested with trypsin to form a single cell suspension and inoculated into cell culture plates (1×10 4 ), cultured in an incubator (37°C, 95% humidity, 5% CO2) for 24 hours, and then treated with glutaric acid for 48 hours. At the same time, HEPES was used to adjust the pH value of the solution to remove the effect of the acidic environment caused by glutaric acid on tumor cells. 4% paraformaldehyde was used to fix the cells at 4°C for 25 minutes, 0.2% Triton X-100 was used for permeabilization for 5 minutes, 1× equilibrium buffer was used for equilibration at room temperature for 20 minutes, 200 μL of labeling reaction solution was added and incubated at 37°C in the dark for 60 minutes, 2 μg / mL of DAPI was used to counterstain the cell nucleus at room temperature for 5 minutes, and PBS was used to wash 3 times, each time for 5 minutes. The distribution of green fluorescence was observed under a fluorescence microscope and images were collected. The results showed that compared with the control group, there was no significant change in the number of green-stained positive cells in HCT116 and MC38 cells treated with glutaric acid, indicating that glutaric acid had no significant effect on the apoptosis of colorectal cancer cells ( Figure 6 ).

[0098] Example 3 In vivo verification of intestinal bacterial metabolite glutaric acid inhibiting colorectal cancer

[0099] (1) Establishing a transplanted tumor model to detect the effect of glutaric acid on mouse tumors

[0100] MC38 cells in the logarithmic growth phase were obtained and digested with trypsin to form a single cell suspension, which was then subcutaneously injected into the upper right thigh of C57BL / 6 mice (8×10 5 ), and when the tumor volume of the mice increased to 100 mm 3Glutaric acid (100 mg / kg) was injected intraperitoneally every two days, and HEPES was used to adjust the pH value of the solution to eliminate the effect of the acidic environment caused by glutaric acid on mice. At the same time, the control group was injected with an equal volume of saline. The model construction process is as follows: Figure 7 As shown in A. During the feeding process, the weight of mice was weighed and recorded weekly. When the tumor volume of mice in the control group increased to 2000mm 3 The tumors of mice in each group were removed and weighed, and the long and short diameters of the tumors were measured with a vernier caliper to calculate the tumor volume. Tumor volume = (long diameter × short diameter) 2 ) / 2. The results showed that compared with the model group, the tumor volume and weight of mice in the glutaric acid treatment group were significantly reduced ( Figure 7 B, C, D), the body weight of mice was not significantly different from that of the model group ( Figure 7 E), indicating that glutaric acid has a significant inhibitory effect on mouse tumors.

[0101] (2) HE staining was used to detect the effect of glutaric acid on mouse tumor tissue

[0102] HE staining was used to detect the effect of glutaric acid on mouse tumor tissue. After dehydration, embedding and sectioning of the tumor tissue, the following steps were performed:

[0103] 1) Dewaxing and hydration: First, bake the slides in a 60°C oven for 30 min, soak them in xylene I and xylene II for 15 min, soak them in anhydrous ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 min, and rinse them with distilled water three times.

[0104] 2) Staining: Stain with hematoxylin for 2 min, rinse with running water for 5 min, dry and then stain with eosin for 10 s, rinse with running water for 5 min.

[0105] 3) Dehydration: Soak in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 2 minutes, and then soak in xylene I and xylene II for 10 minutes.

[0106] 4) Sealing: Use neutral gum to seal the slides, bake at 37°C overnight and then store at room temperature.

[0107] 5) Taking photos: Observe and collect images under a microscope.

[0108] The results showed that compared with the model group, the glutaric acid-treated group had less tumor tissue necrosis, fewer pathological mitotic figures, and weaker proliferation activity, indicating that glutaric acid has a significant inhibitory effect on colorectal cancer ( Figure 8 ).

[0109] (3) Immunohistochemistry was used to detect the expression of Ki67 in tumor tissues

[0110] Immunohistochemistry kit was used to detect the expression of Ki67 in tumor tissues to verify the effect of glutaric acid on tumor cell proliferation in vivo. After dehydration, embedding and sectioning of tumor tissues, the following steps were performed:

[0111] 1) Dewaxing and hydration: First, take out the slices, make a record on the slide and insert them into the slide rack, bake them in a 60°C oven for 30 min, soak them in xylene I and xylene II for 15 min, soak them in 100% ethanol, 90% ethanol, 80% ethanol and 70% ethanol for 5 min, and rinse them with distilled water three times.

[0112] 2) Antigen repair: Place the tissue sections in 200 mL EDTA antigen repair solution and preheat at low temperature for 1 min, high temperature for 2 min, and low temperature for 10 min. Take out the tissue sections and cool them naturally to room temperature. Place the sections in PBS and rinse them three times.

[0113] 3) Removal of endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocker to the sliced ​​tissue, incubate at room temperature for 30 minutes, and rinse three times with PBS, each time for 5 minutes.

[0114] 4) Serum blocking: After the slices are slightly dry, add an appropriate amount of normal goat serum working solution, block at 37°C for 20 minutes, and wash with PBS three times, each time for 3 minutes.

[0115] 5) Incubate with antibodies: Aspirate the surrounding liquid, add an appropriate amount of primary antibody diluent (1:500) to soak the slices, store in a 4°C refrigerator overnight, take out the slices and rewarm for 1 hour, rinse with PBS three times, 5 minutes each time. Incubate with biotin-labeled goat anti-rabbit IgG at 37°C for 20 minutes, rinse with PBS three times, 5 minutes each time. Aspirate the surrounding liquid, add an appropriate amount of horseradish enzyme-labeled streptavidin and incubate for 20 minutes, rinse with PBS three times, 5 minutes each time.

[0116] 6) DAB staining: wipe off the liquid on the slice, add appropriate amount of DAB staining solution, let it stand for staining for 3 minutes, observe under a microscope to avoid over-staining, and wash with tap water for 5 minutes.

[0117] 7) Hematoxylin counterstaining: Hematoxylin counterstaining for 2 minutes, and then washing with distilled water for 3 minutes.

[0118] 8) Dehydration: Soak the tissue sections in 70% ethanol, 80% ethanol, 90% ethanol and anhydrous ethanol for 2 minutes, and then soak them in xylene I and xylene II for 10 minutes.

[0119] The results showed that compared with the model group, the expression of Ki67 in the tumor tissue of the mice in the glutaric acid treatment group was significantly reduced, indicating that glutaric acid has an inhibitory effect on the proliferation of tumor cells in vivo ( Fig. 9 ).

[0120] The above description is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods, and these should also be regarded as the scope of protection of the present invention.

Claims

1. Use of glutaric acid in the preparation of drugs for preventing and / or treating colorectal cancer.

2. The use according to claim 1, characterized in that: Application of glutaric acid in the preparation of a drug that inhibits the proliferation of colorectal cancer cells in a concentration-dependent and time-dependent manner and reduces the proportion of EdU-positive red-stained cells.

3. The use according to claim 1, characterized in that: Application of glutaric acid in the preparation of drugs for inhibiting the migration rate and the number of migrated cells of colorectal cancer cells.

4. The use according to claim 1, characterized in that: Application of glutaric acid in preparing medicine for inhibiting the volume and weight of mouse tumors.

5. The use according to claim 1, characterized in that: Application of glutaric acid in the preparation of medicines for reducing tumor tissue necrosis and the number of pathological mitotic cells.

6. The use according to claim 1, characterized in that: Application of glutaric acid in the preparation of a drug for reducing the expression of Ki67 in mouse tumor tissue.

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