Application of Ackerman bacterium outer membrane protein Amuc1310 in medicine or health food for preventing and treating intestinal cancer

Abstract

The invention belongs to the technical field of postbiotics, and particularly discloses application of Ackermania outer membrane protein Amuc1310 in preparation of drugs or health care products for preventing and treating intestinal cancer. Experimental screening finds that the Amuc1310 protein sourced from Ackerman's bacteria has the effects of inhibiting proliferation of human colorectal adenocarcinoma cells and promoting apoptosis of the human colorectal adenocarcinoma cells, can be used as a beneficial supplement of a current postbiotic treatment means, and provides a new clue and a new thought for prevention and treatment of cancers in the future.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of postbiotics, and specifically discloses application of Akkermansia muciniphila outer membrane protein Amuc_1310 in a drug for preventing and / or treating intestinal cancer or a health food. BACKGROUND

[0002] Colorectal adenocarcinoma is one of the most common digestive tract malignancies worldwide, and its incidence and mortality have long been ranked first among various cancers. The occurrence and development of the disease is a complex process involving multiple stages and multiple factors, and the interaction of various mechanisms such as genetic variation, epigenetic change, chronic inflammation and microenvironment imbalance. Although significant progress has been made in early screening, surgical resection and comprehensive treatment, the long-term prognosis of colorectal adenocarcinoma is still challenged by tumor heterogeneity, drug resistance and metastasis recurrence. In recent years, the role of intestinal microbial homeostasis in the occurrence and development of colorectal adenocarcinoma has attracted increasing attention. Studies have shown that the imbalance of intestinal flora structure and function may indirectly or directly participate in the malignant transformation process of colorectal mucosal epithelium by affecting host immune response, metabolic product generation and intestinal barrier integrity. This phenomenon suggests that maintaining intestinal microbial homeostasis may be an important way to regulate the inflammatory environment of the digestive system and intervene in related malignant transformation.

[0003] Akkermansia muciniphila (Akkermansia muciniphila) Akkermansia muciniphila , AkkProbiotics, as a promising next-generation probiotic, have attracted much attention and become a hot topic in scientific research. However, due to the activity and potential pathogenicity of probiotics, their clinical application faces certain challenges. Metabiotics, on the other hand, refer to preparations of non-living microorganisms or their components that bring health benefits to the host; they are an emerging concept in the field of microecological preparations. Metabiotics are mainly divided into two parts: bacterial cell components and bacterial cell metabolites. Bacterial cell components mainly include membrane proteins, intracellular polysaccharides, peptidoglycans, teichoic acid, lipoteichoic acid, and phosphatal-aldolpids; bacterial cell metabolites include vitamins, lipids, organic acids, bacteriocins, enzymes, extracellular polysaccharides, and short-chain fatty acids. The complex composition determines the diversity of metabiotic functions, including antioxidant, anti-inflammatory, anti-cancer, gut microbiota balancing, enhanced intestinal epithelial barrier function, enhanced immune regulation, and metabolism promotion. Compared with probiotics, metabiotics are more stable, safer, unaffected by antibiotics, have a wider range of targets, and are more easily absorbed by the intestines. Therefore, metabiotics have a wide range of applications, including food, health products, and feed, and have broad market prospects. In Akkermansia bacteria cultured in mucin or glucose media, three outer membrane proteins encoded by the locus tags Amuc_0336, Amuc_1310, and Amuc_1098 showed the highest expression abundance, accounting for approximately 20% to 40% of the total outer membrane proteins. BLAST homology analysis predicted that the 17 kDa protein encoded by the locus tag Amuc_1310 belongs to a protein superfamily containing various rickettsiae-specific 17 kDa surface antigen proteins, suggesting that this protein may be involved in the interaction between the strain and the environment. Currently, there are no reports on the application of Amuc_1310 in the prevention and treatment of intestinal diseases. Summary of the Invention

[0004] The purpose of this invention is to screen for new clinical applications of the Amuc_1310 protein derived from Akkermansia through extensive experiments.

[0005] This invention provides information on the application of Akkermansia outer membrane protein Amuc_1310 in the preparation of drugs or health foods for the prevention and / or treatment of intestinal cancer.

[0006] As a preferred option, the use of Akkermansia outer membrane protein Amuc_1310 in the preparation of drugs or health foods for the prevention and / or treatment of colorectal adenocarcinoma is provided.

[0007] As a preferred embodiment, the drug includes a drug carrier and / or a pharmaceutically acceptable adjuvant therapeutic material; The health food products mentioned include special medical foods, solid beverages, dairy products, soy products, or animal feed.

[0008] The dosage forms of the drug include pills, tablets, capsules, granules, suspensions, or oral liquids; The dosage forms of health food include pills, tablets, capsules, granules, suspensions, oral liquids, enemas, or tube feeding preparations.

[0009] Through experiments, this invention has discovered that the Akkermansia amuc_1310 protein possesses the following properties: (1) Inhibits the proliferation of human colorectal adenocarcinoma cells; (2) Promotes apoptosis of human colorectal adenocarcinoma cells.

[0010] This invention provides the use of the Amuc_1310 protein derived from Akkermansia bacillus for improving human colorectal adenocarcinoma.

[0011] The beneficial effects of this invention are as follows: This invention has shown through experiments that the Amuc_1310 protein derived from Akkermansia can significantly inhibit the proliferation of human colorectal adenocarcinoma cells and promote their apoptosis. It can be used for the prevention and treatment of intestinal cancer, providing new clues and ideas for future cancer prevention and treatment, and can also serve as a beneficial supplement to post-genetic therapy. Attached Figure Description

[0012] Figure 1 The Amuc_1310 protein derived from Akkermansia bacteria in Example 1 inhibits the proliferation of human colorectal adenocarcinoma cells. In the figure, A shows the inhibition of HT29 cell proliferation by different concentrations of Amuc_1310 protein; B shows the inhibition of Caco-2 cell proliferation by different concentrations of Amuc_1310 protein.

[0013] Figure 2 This illustrates how Amuc_1310 protein derived from Akkermansia bacteria promotes apoptosis in human colorectal adenocarcinoma cells, as shown in Example 2. In the figure, A is a flow cytometry scatter plot showing the effect of different concentrations of Amuc_1310 protein on HT29 cell apoptosis; B is a statistical graph showing the total apoptosis rate of different concentrations of Amuc_1310 protein on HT29 cell apoptosis; C is a flow cytometry scatter plot showing the effect of different concentrations of Amuc_1310 protein on Caco-2 cell apoptosis; and D is a statistical graph showing the total apoptosis rate of different concentrations of Amuc_1310 protein on Caco-2 cell apoptosis. Detailed Implementation

[0014] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. Example

[0015] The Akkermansia amuc_1310 protein of this invention was obtained by expression, purification, and large-scale amplification using engineered Escherichia coli BL21(DE3). Detailed steps are as follows: Using pET26b(+)-Amuc_1310 plasmid as a template, primers were designed to amplify the Amuc_1310 gene and clone it into the Pgex4t-1-Amuc_1310 vector.

[0016] Amuc_1310-F:aaaGAATTCATGAAAACAGCTTCCATTCTTACCA Amuc_1310-R:aaaCTCGAGATAGGCCAGCACGCGGGAACCGTTGTT Expression and purification of Amuc_1310 protein: The plasmid containing Pgex4t-1-Amuc_1310 was transformed into BL21(DE3) competent cells, plated on ampicillin-resistant solid medium, and incubated upside down at 37℃ until single colonies grew. Single colonies were picked and transferred to 50 mL of LB liquid medium and incubated at 37℃ and 210 rpm for 8 h. Then, they were transferred to 1000 mL of LB liquid medium and incubated at 37℃ and 165 rpm for 4-6 h until OD (Organic Demand). 600 Reach 0.6-0.8. Add 1 mL of IPTG to a final concentration of 0.5 M to the bacterial culture and induce culture at 18℃ and 165 rpm for 12 h. Collect the bacterial cells by centrifugation at 4℃ and 5000 rpm; resuspend the bacterial cells in 50 mL of Buffer C (200 mM NaCl, 5% glycerol, 10 mM Tri-HCl, pH 8.0), and lyse the cells using an autoclave. Centrifuge the lysed bacterial culture at 12000 rpm and 4℃ for 30 min; add 2 mL of Glutathione beads to each tube of supernatant and bind them using a Ferris wheel for 30 min; add the bound sample to a chromatography column and wash successively with 10-15 column volumes of Buffer C containing 25 mM and 40 mM imidazole, respectively; resuspend the washed liquid in 10 mL of Buffer C containing 10 mM reduced glutathione and collect the protein eluent after 15 min. The eluted protein was concentrated and passed through an AKTA S200 molecular sieve. Western blot was used to verify the purity of the Amuc_1310 protein.

[0017] Example 2: Akkermansia-derived Amuc_1310 protein inhibits the proliferation of human colorectal adenocarcinoma cells. On the first day, cell seeding was performed: HT29 and Caco-2 cell densities were adjusted to 2 × 10⁻⁶ cells using complete culture medium. 4Cells / mL; 100 µL of cell suspension was added to each well of a 96-well plate. On the second day, experimental groups were set up (6 replicates per group): the culture supernatant of each well was discarded; then, the blank group was treated with only complete culture medium (no cells), the control group was treated with 100 µL of complete culture medium, and the experimental groups were treated with different concentrations of Amuc_1310 protein (0.05 / 0.5 / 5 / 50 µg / mL); finally, the 96-well plate was incubated at 37°C in a 5% CO2 incubator for 72 hours. On the fifth day, CCK-8 assay was performed: CCK-8 working solution was prepared by adding 10 µL of CCK-8 reagent + 90 µL of fresh culture medium to each well; the original culture medium was aspirated, and 100 µL of CCK-8 working solution was added to each well; the 96-well plate was returned to the incubator and incubated in the dark for 1 hour; the absorbance (OD value) of each well was measured using a microplate reader at a wavelength of 450 nm.

[0018] Calculate cell viability: ; Plot a cell viability-concentration curve, such as... Figure 1 As shown in Figure A: Compared with the control group, the survival rate of HT29 cells was significantly reduced when the Amuc_1310 protein concentration reached 0.5 μg / mL; compared with the Amuc_1310 protein (0.05 μg / mL) experimental group, the Amuc_1310 protein (0.5 / 5 / 50 µg / mL) significantly reduced the survival rate of HT29 cells. Furthermore, this phenomenon was again verified in Caco-2 cells. Figure 1 B). In summary, Amuc_1310 can inhibit the proliferation of human colorectal adenocarcinoma cells.

[0019] To further verify the effect of Amuc_1310 protein on human colorectal adenocarcinoma cells, cell plating and treatment were performed on day one: the density of HT29 and Caco-2 cells was adjusted to 2 × 10⁶ cells / year. 5 2 mL of the sample was inoculated into each well of a 6-well plate, approximately 4 × 10⁻⁶ cells / mL .... 5Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours to achieve a cell adhesion rate of 60-70%. The next day, protein was added: different concentrations of Amuc_1310 protein solutions (0.05 / 0.5 / 5 / 50 µg / mL) were prepared using complete culture medium; the original culture medium was removed, and the control group received only 2 mL of complete culture medium, while the experimental groups received different concentrations of Amuc_1310 protein (0.05 / 0.5 / 5 / 50 µg / mL). The 96-well plates were then incubated at 37°C in a 5% CO2 incubator for 72 hours. On day 5, cells were collected: they were digested with trypsin without EDTA; the cell suspension was transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; the cells were washed once with pre-chilled PBS, centrifuged, and the supernatant was discarded; 500 µL of 1× Binding Buffer was added to each tube to resuspend the cells; 5 µL of Annexin V-FITC was added, gently mixed, and incubated at room temperature in the dark for 15 minutes; 5 µL of PI was added, mixed, and incubated on ice in the dark for 5 minutes; the cells were analyzed by flow cytometry within 1 hour.

[0020] Calculate the apoptosis rate: Total apoptosis rate (%) = Early apoptosis rate (AV⁺ / PI⁻) + Late apoptosis rate (AV⁺ / PI⁺) The results are as follows Figure 2 As shown in Figures AB: Compared with the control group, the total apoptosis rate of HT29 cells was significantly increased when the Amuc_1310 protein concentration reached 0.5 μg / mL; compared with the Amuc_1310 protein (0.05 μg / mL) experimental group, Amuc_1310 protein (0.5 / 5 / 50 µg / mL) significantly increased the total apoptosis rate of HT29 cells. Furthermore, this phenomenon was again verified in Caco-2 cells. Figure 2 (CD). In summary, Amuc_1310 can promote apoptosis in human colorectal adenocarcinoma cells.

[0021] The above specific embodiments are used to explain and illustrate the present invention, and are only preferred embodiments of the present invention, not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. Use of Akkermansia muciniphila outer membrane protein Amuc_1310 in the preparation of a drug or health food for preventing and / or treating intestinal cancer.

2. Use of Akkermansia muciniphila outer membrane protein Amuc_1310 in the preparation of a drug or health food for preventing and / or treating colorectal adenocarcinoma.

3. Use according to claim 1 or 2, characterized in that: The drug comprises a drug carrier and / or a pharmaceutically acceptable auxiliary treatment material.

4. Use according to claim 1 or 2, characterized in that: The health food comprises a special medical food, a solid beverage, a dairy product, a soy product, or an animal feed.

5. Use according to claim 3, characterized in that: The dosage form of the drug comprises a pill, a tablet, a capsule, a granule, a suspension, or an oral liquid.

6. Use according to claim 4, characterized in that: The dosage form of the health food comprises a pill, a tablet, a capsule, a granule, a suspension, an oral liquid, an enema, or a tube feeding preparation.

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