Application of A. muciniphila bacteria and outer membrane vesicles thereof in relieving chronic stress-mediated colorectal cancer progress

Abstract

The invention belongs to the technical field of biological medicines, and relates to an A.muciniphila bacterium and an application of an outer membrane vesicle of the A.muciniphila bacterium in relieving chronic stress-mediated colorectal cancer progress. The product provided by the invention can effectively improve the influence of chronic stress on the tumor growth of colorectal cancer mice, and specifically comprises the following steps: inhibiting and / or relieving weight loss, relieving intestinal function damage, relieving tumor load increase, reducing tumor cell proliferation activity, and relieving or inhibiting the growth, invasion and metastasis of tumors. The invention provides a new treatment strategy for colorectal cancer patients accompanied by chronic stress.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the mechanism of action of intestinal probiotics and their metabolites in improving chronic stress and promoting the progression of colorectal cancer, and more particularly to the use of Akkermansia muciniphila (A. muciniphila) and its outer membrane vesicles (OMVs) in the treatment of chronic stress-related colorectal cancer. Background Art

[0002] Colorectal cancer is a common malignant tumor of the digestive system. At present, the treatment methods for colorectal cancer include preoperative radiotherapy, surgical local resection, local and metastatic colorectal cancer surgery, endoscopic treatment, systemic treatment, local ablation treatment for metastatic colorectal cancer, specific targeted treatment, and immunotherapy, etc. However, the five-year survival rate of colorectal cancer patients remains low.

[0003] Stress refers to the systemic non-specific adaptive response of the body under the stimulation of internal and external environmental factors, as well as social and psychological stress. Stress can be divided into acute stress and chronic stress (CS). [1] . There is a close association between chronic stress and cancer. Eckerling's research shows that a cancer diagnosis can become a long-term and intense stressor, which in turn leads to persistent psychological distress in patients. [2] . Similarly, epidemiological studies have shown that compared with the general population, cancer patients face a greater burden of mental health disorders, and the incidence of anxiety and depression is significantly increased. [3,4] .

[0004] A large number of symbiotic microbial communities (Gut microbiota, GM) are colonized in the body's intestine, including bacteria, fungi, viruses, archaea, and parasites, etc. The gut microbiota plays an important role in maintaining the homeostasis of the host intestinal environment, such as metabolizing nutrients, regulating the host immune capacity, and participating in host defense. However, the microbial community in the intestine is dynamic and is easily affected by various factors, including diet, drugs, mental stress, etc. An animal study has shown that chronic stress changes the composition and metabolic function of the gut microbiota in rats. [5] . In addition, another study pointed out that chronic stress is associated with a decrease in the richness and diversity of the gut microbial community. [6] . Research has shown that dysbiosis is associated with the occurrence and development of various malignant tumors, including colorectal cancer, gastric cancer, hepatocellular carcinoma, breast cancer, pancreatic cancer, lung cancer, etc. [7-12] . Since the gut microbiota is in direct contact with colorectal cancer cells, a disordered gut microbiota is particularly important in the progression of colorectal cancer.

[13] . Research by Suuny H has shown that transplanting the feces of colorectal cancer patients can significantly increase the number of tumors in germ-free colorectal cancer mice compared to the feces of healthy individuals.

[14] .

[0005] The typical feature of dysbiosis is a reduction in gut microbial diversity or a change in resident species. Dysregulated gut microbes can be classified into probiotics, neutral bacteria, and harmful bacteria. It has been reported that gut probiotics inhibit the occurrence of colorectal cancer by releasing specific proteins and metabolites. However, there is currently no intervention strategy for chronic stress to regulate CRC progression through the gut microbiota.

[0006] Cited references:

[0007] [1] DAI S, MO Y, WANG Y, et al. Chronic Stress Promotes Cancer Development[J]. Front Oncol, 2020, 10: 1492.

[0008] [2] ABEDIZADEH R, MAJIDI F, KHORASANI H R, et al. Colorectal cancer: a comprehensive review of carcinogenesis, diagnosis, and novel strategies for classified treatments[J]. Cancer Metastasis Rev, 2024, 43(2): 729 - 53.

[0009] [3] HARTUNG T J, E, FALLER H, et al. The risk of being depressed is significantly higher in cancer patients than in the general population: Prevalence and severity of depressive symptoms across major cancer types[J]. Eur J Cancer, 2017, 72: 46 - 53.

[0010] [4] MITCHELL AJ, CHAN M, BHATTI H, et al. Prevalence of depression, anxiety, and adjustment disorder in oncological, haematological, and palliative-care settings: a meta-analysis of 94 interview-based studies[J]. Lancet Oncol, 2011, 12(2): 160 - 74.

[0011] [5] XU M, WANG C, KROLICK KN, et al. Difference in post-stress recovery of the gut microbiome and its altered metabolism after chronic adolescent stress in rats[J]. Sci Rep, 2020, 10(1): 3950.

[0012] [6] BAILEY MT, DOWD SE, GALLEY JD, et al. Exposure to a social stressor alters the structure of the intestinal microbiota: implications for stressor-induced immunomodulation[J]. Brain Behav Immun, 2011, 25(3): 397 - 407.

[0013] [7] DOUGHERTY MW, JOBIN C. Intestinal bacteria and colorectal cancer: etiology and treatment[J]. Gut Microbes, 2023, 15(1): 2185028.

[0014] [8] GOTO T. Microbiota and lung cancer[J]. Semin Cancer Biol, 2022, 86(Pt3): 1 - 10.

[0015] [9]HERRERA-QUINTANAL, VáZQUEZ-LORENTE H, PLAZA-DIAZ J. Breast Cancer: Extracellular Matrix and Microbiome Interactions[J]. Int J Mol Sci, 2024, 25(13).

[0016]

[10] LIU Y, BABA Y, ISHIMOTO T, et al. Gut microbiome in gastrointestinal cancer: a friend or foe?[J]. Int J Biol Sci, 2022, 18(10): 4101-17.

[0017]

[11] RATTAN P, MINACAPELLI C D, RUSTGI V. The Microbiome and Hepatocellular Carcinoma[J]. Liver Transpl, 2020, 26(10): 1316-27.

[0018]

[12] SEXTON R E, UDDIN M H, BANNOURA S, et al. Correction to: Connecting the human microbiome and pancreatic cancer[J]. Cancer Metastasis Rev, 2022, 41(2): 333.

[0019]

[13] WONG C C, YU J. Gut microbiota in colorectal cancer development and therapy[J]. Nat Rev Clin Oncol, 2023, 20(7): 429-52.

[0020]

[14] WONG S H, ZHAO L, ZHANG X, et al. Gavage of Fecal Samples From Patients With Colorectal Cancer Promotes Intestinal Carcinogenesis in Germ-Free and Conventional Mice[J]. Gastroenterology, 2017, 153(6): 1621-33.e6. SUMMARY OF THE INVENTION

[0021] Problems to be Solved by the Invention

[0022] Currently, there have been relevant reports on the treatment of A. muciniphila in chronic stress or cancer, but there is no relevant report on its role in the progression of colorectal cancer mediated by chronic stress. The inventors found through research that colorectal cancer patients usually have emotional disorders such as anxiety and depression after being diagnosed with colorectal cancer, which exacerbate the symptoms of colorectal cancer or accelerate the progression of colorectal cancer, and are not conducive to the treatment of colorectal cancer. Therefore, the present invention constructs a colorectal cancer + chronic stress model in mice to simulate the physiological and psychological states of colorectal cancer patients, and thus finds a method that can effectively relieve or inhibit the promotion of colorectal cancer progression caused by chronic stress such as emotional disorders like anxiety and depression, providing a new strategy for the treatment of chronic stress-related colorectal cancer.

[0023] Solutions for Solving the Problems

[0024] [1]. Use of Akkermansia muciniphila and / or its outer membrane vesicles in the preparation of a product for relieving and / or inhibiting the progression of chronic stress-mediated colorectal cancer.

[0025] [2]. The use according to [1], wherein the relieving and / or inhibiting the progression of chronic stress-mediated colorectal cancer includes at least one of (a) to (e):

[0026] (a) Relieving and / or inhibiting the promoting effect of chronic stress on the weight loss of the body suffering from colorectal cancer;

[0027] (b) Relieving and / or inhibiting the promoting effect of chronic stress on the intestinal function injury of the body suffering from colorectal cancer;

[0028] (c) Relieving and / or inhibiting the promoting effect of chronic stress on the tumor burden in the body suffering from colorectal cancer;

[0029] (d) Relieving and / or inhibiting the promoting effect of chronic stress on the proliferation activity of colorectal cancer tumor cells;

[0030] (e) Relieving and / or inhibiting the promoting effect of chronic stress on the pathological progression of colorectal cancer tumor tissue.

[0031] Optionally, the relieving and / or inhibiting the progression of chronic stress-mediated colorectal cancer further includes improving chronic stress-mediated emotional disorders, and the emotional disorders include depression and / or anxiety.

[0032] [3]. Use according to [2], wherein the tumor burden includes the total number of tumors, tumor volume, and / or the number of tumors greater than 4 mm.

[0033] [4]. Use according to [2] or [3], wherein the histopathological progression of the tumor includes tumor growth, invasion, and metastasis.

[0034] [5]. Use according to any one of [2] to [4], wherein the organism includes mammals;

[0035] Optionally, the mammals include mice, rats, guinea pigs, cows, sheep, cats, dogs, horses, rabbits, pigs, monkeys, and humans.

[0036] [6]. Use according to any one of [1] to [5], wherein the product contains one or more of live cells of Akkermansia muciniphila, cell cultures, and cell culture supernatants.

[0037] [7]. Use according to any one of [1] to [6], wherein the product includes a drug and / or a pharmaceutical composition.

[0038] [8]. Use according to [7], wherein the pharmaceutical composition further contains (i) and / or (ii):

[0039] (i) one or more pharmaceutically acceptable carriers;

[0040] (ii) one or more drugs for clinically treating colorectal cancer.

[0041] [9]. Use according to [8], wherein the pharmaceutically acceptable carrier includes one or a combination of two or more of a solvent, solubilizer, cosolvent, emulsifier, flavoring agent, odorant, coloring agent, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, pH regulator, stabilizer, surfactant, and preservative.

[0042]

[10] . Use according to any one of [7] to [8], wherein the pharmaceutical composition is formulated into a solid preparation, semi-solid preparation, or liquid preparation.

[0043] Effects of the Invention

[0044] The product containing Akkermansia muciniphila and / or its outer membrane vesicles provided by the present invention can significantly improve the effect of chronic stress on tumor growth in colorectal cancer mice, specifically including: inhibiting and / or alleviating weight loss, alleviating intestinal function damage, alleviating increased tumor burden, reducing tumor cell proliferation activity, and alleviating or inhibiting tumor growth, invasion, and metastasis. It further reveals the potential role of A. muciniphila in the treatment of colorectal cancer, providing a new treatment strategy for colorectal cancer patients accompanied by chronic stress. Description of the Drawings

[0045] Figure 1 : Role of gut microbiota in chronic stress-mediated colorectal cancer tumor growth; wherein, A: Representative images of tumor tissues; B: Number of tumors; C: Tumor volume; D: Tumor size; * Indicates P < 0.05, ** Indicates P < 0.01, *** Indicates P < 0.001; CAC represents the colorectal cancer model, CAC+Stress represents the colorectal cancer combined with chronic stress model, and CAC+Stress+FMT represents the treatment with fecal microbiota transplantation after establishing the colorectal cancer combined with chronic stress model.

[0046] Figure 2 : Effect of chronic stress on the abundance of A. muciniphila in tumor tissues; wherein, A: Changes in the abundance of A. muciniphila in mouse tumor tissues; B: Correlation analysis of the abundance of A. muciniphila in mice; C: Changes in the abundance of A. muciniphila in patient tumor tissues; D: Correlation analysis of the abundance of A. muciniphila in patients; * Indicates P < 0.05, ** Indicates P < 0.01, *** Indicates P < 0.001; CAC represents the colorectal cancer model, CRC represents colorectal cancer, and Stress represents the chronic stress model.

[0047] Figure 3 : Evaluation of A. muciniphila colonization; wherein, A: Relative quantitative PCR of A. muciniphila; *** Indicates P < 0.001.

[0048] Figure 4 : Results of open field and tail suspension tests of mice in each group after 14 days of A. muciniphila intervention; A: Results of open field test; B: Results of tail suspension test. *Indicates P < 0.05, **Indicates P < 0.01, ***Indicates P < 0.001.

[0049] Figures 5A to 5D:Results of open field and tail suspension tests in mice of each group after 14 days of intervention with A. muciniphila OMVs; Figure 5A :Results of nanoparticle tracking analysis; Figure 5B :Results of transmission electron microscopy; Figure 5C :Results of open field test; Figure 5D :Results of tail suspension test; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0050] Figure 6 :Effects of A. muciniphila on body weight and disease activity index in mice with chronic stress-induced colorectal cancer; Note: A. Body weight change rate; B. Disease activity index; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0051] Figure 7 :Effects of A. muciniphila on tumor burden in chronic stress-mediated colorectal cancer; A: Representative images of tumor tissues; B: Number of tumors; C: Tumor volume; D: Tumor size; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0052] Figure 8 :Effects of A. muciniphila on tumor cell proliferation in chronic stress-mediated colorectal cancer; A: Representative images of tissue sections; B: Ratio of Ki67-positive area in tissue sections; * indicates P < 0.05, ** indicates P < 0.01.

[0053] Figure 9 :Effects of A. muciniphila on pathological progression of tumor tissues in chronic stress-mediated colorectal cancer; A: Representative images of tissue sections; B: Histopathological score of tissue sections; * indicates P < 0.05, ** indicates P < 0.01.

[0054] Figure 10 :Effects of A. muciniphila OMV on body weight and disease activity index in mice with chronic stress-induced colorectal cancer; Among them, A: Body weight change rate; B: Disease activity index; * indicates P < 0.05.

[0055] Figure 11:Effect of A.muciniphila OMV on colorectal cancer tumor burden mediated by chronic stress; wherein, A: representative images of tumor tissues; B: number of tumors; C: tumor volume; D: tumor size; * Indicates P < 0.05, ** Indicates P < 0.01.

[0056] Figure 12 :Effect of A.muciniphila OMV on the proliferation of colorectal cancer tumor cells mediated by chronic stress; wherein, A: representative images of tissue sections; B: ratio of Ki67 positive area in tissue sections; ** Indicates P < 0.01.

[0057] Figure 13 :Effect of A.muciniphila OMV on the pathological progression of colorectal cancer tumor tissues mediated by chronic stress; wherein, A: representative images of tissue sections; B: histopathological score of tissue sections; * Indicates P < 0.05, *** Indicates P < 0.001.

[0058] Figure 14 :Schematic diagram of A.muciniphila and A.muciniphila OMV improving the effect of chronic stress on colorectal cancer. Detailed implementation manners

[0059] The various exemplary embodiments, features and aspects of the present invention will be described in detail below. The special word "exemplary" here means "serving as an example, embodiment or illustration". Any embodiment described as "exemplary" here does not have to be construed as superior or better than other embodiments.

[0060] In addition, in order to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In other instances, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0061] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0062] In the present invention, the meaning expressed by using "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0063] In the present invention, the "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc. refer to the specific elements (e.g., features, structures, properties, and / or characteristics) related to the embodiments, which are included in at least one of the embodiments described herein, and may or may not exist in other embodiments. Additionally, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0064] In the present invention, "optional" and "optionally" mean that the subsequent described event or situation may or may not occur, and the description includes the situation where the event or situation occurs and the situation where the event or situation does not occur.

[0065] In the present invention, the numerical range represented by "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0066] In the present invention, the term "and / or" encompasses all combinations of the items connected by this term, and should be regarded as each combination having been individually listed herein. For example, "A and / or B" encompasses "A", "A and B", and "B". For example, "A, B, and / or C" encompasses "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0067] In the present invention, "Akkermansia muciniphila", "Akkermansia muciniphila", "A. muciniphila", "Akk", "Akk bacteria" have the same meaning and can be used interchangeably. It refers to a Gram-negative anaerobic bacterium belonging to the phylum Verrucomicrobia, which is mainly colonized in the intestinal mucosal layer of humans and animals and is a normal flora of the human intestine. Akkermansia muciniphila is colonized in the human intestine at different growth stages. In infancy, Akkermansia muciniphila can be used as a marker for the growth status and diversity of the intestinal flora. During the process from early life to adulthood, the number of Akkermansia muciniphila increases significantly.

[0068] In the present invention, "OMV", "outer membrane vesicles of bacteria" refer to bilayer lipid nanovesicles secreted by Gram-negative bacteria. OMVs contain various derived components of bacteria, mainly including lipids, proteins, nucleic acids, and other small molecules. The OMV membrane mainly contains phospholipids (PL) and lipopolysaccharides (LPS). PL mainly includes phosphatidylethanolamine, phosphatidylglycerol, etc. The phospholipid content of OMVs from different Gram-negative bacteria varies.

[0069] In addition, unless otherwise defined, other technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0070] Products Containing Akkermansia muciniphila

[0071] In some aspects of the present invention, the product contains one or more of live cells of Akkermansia muciniphila, cell cultures, cell culture supernatants, and outer membrane vesicles.

[0072] In some embodiments, the above-mentioned live cells, cell cultures, cell culture supernatants, and outer membrane vesicles can all be obtained by conventional culture and treatment methods in the art. The culture of the cells of the present invention can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, fed-batch culture, etc., and various culture conditions such as temperature, time, and pH value of the culture medium can be appropriately adjusted according to the actual situation.

[0073] In some exemplary embodiments, the postbiotic composition described in the present invention is prepared according to the following process: (a) preparing a seed solution of Akkermansia muciniphila; (b) inoculating the seed solution of Akkermansia muciniphila into a culture medium; (c) culturing the Akkermansia muciniphila for a predetermined amount of time to produce a culture, that is, obtaining a cell culture. The cell culture is centrifuged at a low speed, such as 4000-5000 g, the precipitated part is live cells, and the supernatant part is the cell culture supernatant.

[0074] In some specific embodiments, the cell culture supernatant is further centrifuged at a centrifugation speed of >10000 g (such as 15000 g), the supernatant is taken and impurities are removed using a 0.45 μm filter, the filtered filtrate is centrifuged at a higher centrifugation speed (such as >15000 g, 17000 g) to take the precipitate, and the precipitate is purified to obtain the outer membrane vesicles of Akkermansia muciniphila.

[0075] In some embodiments, for the convenience of preservation and product preparation, operations such as heat treatment, physical treatment, spray drying, or freeze drying can also be performed on the above-mentioned live cells, cell cultures, cell culture supernatants, outer membrane vesicles, etc.

[0076] In some embodiments, the Akkermansia muciniphila is currently deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany, Inhoffenstrasse, with the deposit number DSM No. 22959, and anyone can obtain this strain through commercial channels.

[0077] In some embodiments, the content of Akkermansia muciniphila in the product is not less than 1×106 CFU / g or 1×10 6 CFU / mL, for example, it can be 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 CFU / g, 1×10 10 CFU / g, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL, etc.

[0078] In some embodiments, the concentration of outer membrane vesicles in the product is 10 to 500 μg / mL, preferably 25 to 200 μg / mL, more preferably about 50 μg / mL, about 100 μg / mL or about 200 μg / mL (expressed as the total protein in the vesicles).

[0079] In some embodiments, the product comprises a drug or a pharmaceutical composition.

[0080] In some alternative embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

[0081] In some embodiments, the pharmaceutically acceptable carrier includes one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, flavoring agents, odor-masking agents, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants and preservatives.

[0082] In some embodiments, the pharmaceutical composition is formulated into solid preparations, semi-solid preparations or liquid preparations. Among them, the applicable forms of solid preparations mainly include tablets, capsules, granules, pills, etc.; the applicable forms of semi-solid preparations mainly include gels, suppositories, ointments, etc.; the applicable forms of liquid preparations mainly include aerosols, emulsions, mixtures, suspensions, solutions, injections, syrups, tinctures, etc.

[0083] In some preferred embodiments, the pharmaceutical composition is formulated into oral preparations or inhalation preparations, including aerosols, suspensions, solutions, syrups, tinctures, tablets, capsules, granules, pills, etc.

[0084] Use for Relieving and / or Inhibiting the Progression of Chronic Stress-Mediated Colorectal Cancer

[0085] In some aspects of the present invention, administering Akkermansia muciniphila or outer membrane vesicles of Akkermansia muciniphila to a mouse model of colorectal cancer + chronic stress can improve the mood disorders of mice with colorectal cancer caused by chronic stress, and at the same time alleviate or inhibit the progression of colorectal cancer mediated by chronic stress.

[0086] In some specific embodiments, the alleviation of depressive-like and / or anxiety-like behaviors includes a significant reduction in the immobility time and a significant increase in the movement distance of depressed and anxious mice in the open field test and the tail suspension test.

[0087] Furthermore, the alleviation and / or inhibition of the progression of colorectal cancer mediated by chronic stress includes at least one of (a) to (e):

[0088] (a) Alleviating and / or inhibiting the promotion of weight loss in an organism suffering from colorectal cancer.

[0089] In some embodiments, the trend of weight loss in the organism is inhibited, and a trend of weight gain is presented.

[0090] (b) Alleviating and / or inhibiting the promotion of intestinal function damage in an organism suffering from colorectal cancer.

[0091] (c) Alleviating and / or inhibiting the promotion of tumor burden in an organism suffering from colorectal cancer by chronic stress.

[0092] In some embodiments, the tumor burden includes the total number of tumors, tumor volume, and / or the number of tumors larger than 4 mm. In some specific embodiments, the total number of tumors, tumor volume, and the number of tumors larger than 4 mm are all significantly reduced.

[0093] (d) Alleviating and / or inhibiting the promotion of cell proliferation activity in colorectal cancer tumor tissue by chronic stress.

[0094] In some specific embodiments, the expression level of Ki67 is significantly reduced; Ki67 is a cell proliferation marker that is mainly expressed in the G1, S, G2, and M phases of the cell cycle but not in the G0 (quiescent) phase, so the expression level of Ki67 can reflect the proliferation activity of cells.

[0095] (e) Alleviating and / or inhibiting the promotion of the pathological progression of colorectal cancer tumor tissue by chronic stress.

[0096] In some embodiments, the pathological progression of the tumor tissue includes tumor growth, invasion, and metastasis. In some specific embodiments, the histopathological score of the tumor tissue is significantly reduced.

[0097] In some embodiments, the organism includes mammals; further, the mammals include mice, rats, guinea pigs, cows, sheep, cats, dogs, horses, rabbits, pigs, monkeys, and humans.

[0098] Method for Relieving and / or Inhibiting the Progression of Chronic Stress-Mediated Colorectal Cancer

[0099] In some aspects of the present invention, the present invention provides a method for alleviating and / or inhibiting the progression of chronic stress-mediated colorectal cancer, the method comprising administering to a subject in need thereof an effective amount of the product comprising Akkermansia muciniphila described above.

[0100] The subject can be a mammal, including, for example, a human subject in need of treatment for a specific disease (e.g., colorectal cancer, mood disorder caused by colorectal cancer, chronic stress-mediated colorectal cancer progression).

[0101] In some specific embodiments, when the above product is administered to a subject, the therapeutically effective amount for effecting a change or producing a therapeutic effect in the subject can be about 1×10 8 CFU or more, and in some cases, about 1×10 9 、1×10 10 、1×10 11 、1×10 12 or 1×10 13 CFU or more.

[0102] In other embodiments, when the above product is administered to a subject, the therapeutically effective amount for effecting a change or producing a therapeutic effect in the subject can be about 10 μg / mL (expressed as the total protein in the vesicles) or more, and in some cases, about 50 μg / mL, about 100 μg / mL, or about 200 μg / mL or more.

[0103] Examples

[0104] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0105] (I) Materials and methods:

[0106] 1. Establishment of a colorectal cancer model

[0107] A colorectal cancer model was established using Aom combined with DSS solution. The treatment protocol was as follows: On day 0, mice were intraperitoneally injected with 10 mg / kg Aom solution. On day 7, they were allowed to freely drink 2% DSS solution. On day 14, they switched to drinking pure water. On day 28, they drank 2% DSS solution again. On day 35, they switched to drinking pure water. On day 49, they drank 2% DSS solution again and then switched to drinking pure water.

[0108] 2. Establishment of chronic stress model

[0109] In the present invention, chronic stress was induced in mice in a mild, long-term and unpredictable manner. The stressors used included (1) a cage without bedding (6 h); (2) a cage with water (6 h); (3) a dirty cage with an unpleasant odor (6 h); (4) wet bedding (6 h); (5) tilted cage position (tilted at 45°, 24 h); (6) noise stimulation (15 min); (7) foot shock (200 μA, randomly performed 20 times within 30 min, each lasting 1 s); (8) swimming in ice water (15 °C, 5 min); (9) chronic restraint (50 ml centrifuge tube, 6 h); (10) inversion of the 12:12 h light-dark cycle. The entire stress process lasted for 14 days.

[0110] 3. Establishment of a colorectal cancer mouse model with chronic stress

[0111] After establishing a colorectal cancer model using Aom combined with Dss solution, chronic stress was induced in mice in a mild, long-term and unpredictable manner by the means of establishing the chronic stress model described above, thus obtaining a colorectal cancer mouse model with chronic stress.

[0112] 4. Source of A. muciniphila

[0113] The stock solution of A. muciniphila was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. The product name is Akkermansia muciniphila (AKK bacteria), and the product number is B336076. This strain is currently deposited in the DSMZ - German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany, at the deposit number DSM No. 22959. A. muciniphila was cultured for 72 h under anaerobic conditions at 37 °C to obtain the bacterial solution.

[0114] 5. Extraction and determination of A. muciniphila OMV

[0115] The A. muciniphila culture solution was centrifuged at 4°C and 5000g for 10 min to collect the A. muciniphila supernatant. Subsequently, the supernatant was centrifuged at 4°C and 15,000g for 20 min. After centrifugation, the supernatant was taken and a 0.45 μm filter was used to remove impurities such as bacterial particles in the supernatant. Then, the filtered solution was ultracentrifuged at 4°C and 170,000g for 80 min to collect the precipitate. After resuspending the precipitate with sterile PBS, it was centrifuged again at 4°C and 170,000g for 60 min to purify A. muciniphila OMV. After the ultracentrifugation was completed, the precipitate was resuspended with sterile PBS on a clean bench and filtered through a 0.45 μm filter to obtain a sterile A. muciniphila OMVs solution. Finally, the solution was stored in a -80°C refrigerator for subsequent use. Nanoparticle tracking analysis and transmission electron microscopy were used to verify its particle size (50 - 200 nm) and integrity.

[0116] 6. 16S rRNA sequencing

[0117] In the present invention, 16S rRNA sequencing was used to detect the changes in the flora in the mouse intestine. The treatment protocol was as follows: After the behavioral test was completed, the experimental mice were euthanized and fresh intestinal contents were collected for 16S rRNA sequencing analysis.

[0118] 7. Determination of the abundance of A. muciniphila in feces and tumor tissues

[0119] In the present invention, relative quantitative PCR was used to detect the abundance of A. muciniphila in feces. Primers used for relative quantification:

[0120] 1. A. muciniphila - F1 (SEQ ID NO:1): 5'-CAGCACGTGAAGTGGGAC-3';

[0121] 2. A. muciniphila - R1 (SEQ ID NO:2): 5'-CCTTGCGGTTGGCTTCAGAT-3'.

[0122] Reference gene and primers used for amplifying the reference gene:

[0123] 3. Fecal internal control primer F2 (SEQ ID NO:3): 5'-CGGCAACGAGCGCAACCC-3';

[0124] 4. Fecal internal control primer R2 (SEQ ID NO:4): 5'-CCATTGTAGCACGTGTGTAGCC-3'.

[0125] The abundance of A. muciniphila in tumors was detected by absolute quantitative PCR. The treatment protocol is as follows:

[0126] (1) After obtaining tumor tissues from colorectal cancer mice and clinical colorectal cancer patients, they were immediately frozen in an -80 °C refrigerator.

[0127] (2) Nucleic acid molecules were extracted from the tissues, the target product was ligated to the vector pUC57, and quality inspection was carried out. After passing the quality inspection, the formal experiment began.

[0128] (3) According to the concentration of the standard product and the molecular weight (MW) of the plasmid, the copy concentration was calculated, in copies / μl.

[0129] (4) The concentration of the standard quality plasmid after dissolution was 100 ng / μl. The plasmid was diluted in eight gradients and diluted according to the copy number in the series of 10 2 ,10 3 ,10 4 ,10 5 ,10 6 ,10 7 ,10 8 ,10 9 copies / μl; 3 parallel PCR reactions were carried out for each gene of each standard quality plasmid and sample. The reaction mixture was prepared according to the reaction system, shaken and centrifuged, and dispensed into the PCR plate. For each sample and each gene, 3 parallel PCR reactions were performed, and then the corresponding 1 μl of DNA sample or standard product was added. The sealing film was carefully adhered and sealed tightly, shaken and centrifuged, and the prepared PCR plate was placed on ice before setting the PCR program.

[0130] (5) The absolute quantitative data analysis method was used to calculate the copy number of the target gene in the unit amount.

[0131] 8. Treatment of A. muciniphila in mice

[0132] The A. muciniphila administration protocol is as follows: Experimental mice (weighing 20 - 22 g) were orally administered 200 μl of A. muciniphila bacterial solution (1×10 9(CFU), administered continuously for 14 days. (For the setting of the dosage and the administration method, refer to the literature: L. Fan, C. Xu, Q. Ge, Y. Lin, C. C. Wong, Y. Qi, B. Ye, Q. Lian, W. Zhuo, J. Si, S. Chen, L. Wang, A. Muciniphila Suppresses Colorectal Tumorigenesis by Inducing TLR2 / NLRP3-Mediated M1-Like TAMs. Cancer Immunol Res 9, 1111-1124 (2021).)

[0133] 9. Treatment of mice with A. muciniphila OMV

[0134] The administration protocol of A. muciniphila OMV is as follows: Experimental mice (weighing 20-22 g) were orally administered 100 μl of A. muciniphila OMV suspension (containing 200 μg / mL, expressed as the total protein in the vesicles) every day for 14 consecutive days. (For the setting of the dosage and the administration method, refer to the literature: X. Wang, S. Lin, L. Wang, Z. Cao, M. Zhang, Y. Zhang, R. Liu, J. Liu, Versatility of bacterial outer membrane vesicles in regulating intestinal homeostasis. Sci Adv 9, eade5079 (2023).)

[0135] 10. Open field test

[0136] The open field test was used to detect the anxiety of mice. Three days before the start of the behavioral experiment, the mice were placed in the behavioral laboratory for 30 min every day to adapt to the environment. During the test phase, the experimental environment was kept quiet, and the mice were placed with their backs to the experimenter at the center of the open field box from a fixed position. The movement trajectories of each mouse for 5 min were recorded using an automatic activity monitoring system (ANY-maze software). The total moving distance, average moving speed, number of entries into the central area, and moving distance in the central area of each mouse in the open field box were statistically analyzed.

[0137] 11. Tail suspension test

[0138] The tail suspension test was used to detect the depressive mood of mice. Three days before the start of the behavioral experiment, the mice were placed in the behavioral laboratory for 30 minutes every day to adapt to the environment. During the test phase, the experimental environment was kept quiet, and the tails of the mice were fixed to the top of the tail suspension box, allowing them to struggle freely for 6 minutes. The movement trajectories in the last 4 minutes were recorded using an automatic activity monitoring system (DigBehv Animal Behavior Video Analysis System). The immobility time and the percentage of immobility time of the experimental mice in the tail suspension box were statistically analyzed.

[0139] 12. General status observation of mice

[0140] The body weight, fecal traits, and intestinal bleeding of the mice were recorded daily until the end of the experiment. The calculation formula for the body weight change rate: (body weight on the current day / original body weight) × 100%. The calculation formula for the body weight loss ratio: ((body weight on the current day - original body weight) / original body weight) × 100%. The disease activity index includes three parts: body weight loss ratio, fecal traits, and intestinal bleeding, which is used to evaluate the degree of intestinal function damage. The scoring criteria are shown in Table 1 for details.

[0141] Table 1. Scoring criteria for disease activity index

[0142]

[0143] 13. Histopathological progression of tumor tissues

[0144] HE staining was used to detect the histopathological scoring of tumors in mice of each group. The scoring criteria are as follows: Three fields of view were taken from each slide, and after each field of view was scored according to the following criteria, the sum of the scores was taken.

[0145] Scores per Field of View Score Normal Tissue 0 Presence of Inflammation or Absence of Intestinal Tissue 1 Slight Nuclear Enlargement, Nuclear Crowding, Nuclear Hyperchromasia, and Decrease or Loss of Intracellular Mucin 2 Prominent Nuclear Stratification, Severe Nuclear Hyperchromasia or Pleomorphism, and Obvious Structural Distortion 3 Back-to-Back Glands, Absence of Stroma, Epithelial Cell Dysplasia, and Invasion of the Colon Base 4

[0146] 14. Colony formation assay to detect the proliferation ability of tumor cells

[0147] Cells were seeded in 6-well plates. When the cell density reached about 70%, 20 μg of A. muciniphila OMV was added to the cells and cultured for 48 hours, and then replaced with normal medium and continued to be cultured for 14 days. The cell colonies were fixed with methanol for 10 minutes and stained with 0.1% crystal violet at room temperature for 15 minutes. The cell colonies were counted using ImageJ software.

[0148] 15. Fecal microbiota transplantation

[0149] The present invention uses fecal microbiota transplantation to restore the intestinal flora of mice. The treatment scheme is as follows: collect fresh feces from the control group mice and place them in a pre-cooled sterile tube. Add sterile PBS at a ratio of 100 mg / ml and fully homogenize. Use a filter to filter the fecal suspension to remove fecal particles. Centrifuge at 4°C and 500g for 3 minutes, and collect the supernatant. After centrifugation again, prepare a fecal microbiota suspension and store it in a -80°C refrigerator for use. The recipient mice are gavaged with 200ul of fecal microbiota suspension every day to restore the intestinal flora.

[0150] (II) Results:

[0151] 1. Role of intestinal flora in chronic stress-mediated colorectal cancer tumor growth

[0152] The gross observation results of colon tissue after fecal microbiota transplantation in chronically stressed colorectal cancer mice are as follows: Figure 1 As shown, the number, volume, and number of tumors larger than 4 mm in the CAC+Stress+FMT group were significantly reduced compared with those in the CAC+Stress group (P < 0.001, P < 0.01, P < 0.01). These results suggest that the presence of certain beneficial bacteria in the intestinal flora may play an important role in improving the progression of colorectal cancer mediated by chronic stress. Through database screening, the inventors found that A. muciniphila may be a potential probiotic for alleviating the progression of colorectal cancer mediated by chronic stress.

[0153] 2. Effect of chronic stress on the abundance of A. muciniphila in tumor tissues

[0154] To investigate the abundance of A. muciniphila in colorectal cancer tissues with chronic stress, we used absolute quantitative PCR to detect A. muciniphila in colorectal cancer mice and patient tumor tissues. Figure 2 As shown in the results, compared with the non-stressed group, the abundance of A. muciniphila in the tumor tissues of colorectal cancer mice and patients in the stressed group was significantly reduced (P < 0.001, P < 0.001). In addition, correlation analysis showed that the abundance of A. muciniphila was negatively correlated with the tumor volume (P < 0.01, P < 0.05). These results indicate that chronic stress can reduce the abundance of A. muciniphila in tumor tissues.

[0155] 3. Assessment of A. muciniphila colonization

[0156] After chronic stress colorectal cancer mice were gavaged with A. muciniphila for 14 days, the results of relative quantitative PCR showed that ( Figure 3) Compared with the CAC+Stress group, the abundance of A. muciniphila in the feces of the CAC+Stress+A. muciniphila group was significantly increased (P < 0.001). This result indicates that A. muciniphila successfully colonized the intestine of mice with chronic stress-induced colorectal cancer.

[0157] 4. A. muciniphila alleviates anxiety and depressive-like behaviors in mice with stress-induced colorectal cancer

[0158] The anxiety-like behaviors of mice were detected by the open-field test. The results Figure 4 showed that compared with the mice in the CAC group (9.165 ± 3.167), the exploration distance of the mice in the Stress+CAC group (5.854 ± 1.894) in the open-field chamber was significantly shortened (t = 2.196, P = 0.0161); after treatment with A. muciniphila, compared with the mice in the Stress+CAC group (5.854 ± 1.894), the exploration distance of the mice in the A. muciniphila+Stress+CAC group (11.22 ± 2.737) was significantly increased (t = 4.747, P = 0.0003)( Figure 4 A). The depressive-like behaviors of mice were detected by the tail suspension test, and it was found that the immobility time of the mice in the Stress+CAC group (164.1 ± 47.29) was significantly increased compared with that of the mice in the CAC group (126.9 ± 18.87) (t = 2.191, P = 0.0436). However, the immobility time of the mice with chronic stress-induced colorectal cancer after A. muciniphila intervention (108.8 ± 53) was significantly reduced (t = 2.27, P = 0.0381)( Figure 4 B). The above results indicate that A. muciniphila can alleviate anxiety and depressive-like behaviors in mice with stress-induced colorectal cancer.

[0159] 5. A. muciniphila OMVs alleviate anxiety and depressive-like behaviors in mice with stress-induced colorectal cancer

[0160] Gram-negative anaerobic bacteria usually release parental outer membrane vesicles (OMVs) during growth, which contain various native components such as nucleic acids, proteins, enzymes, and lipopolysaccharides. After the inventors isolated OMVs from the supernatant of A. muciniphila culture, the isolated A. muciniphila OMVs were characterized by NTA and TEM. As Figure 5A and Figure 5BAs shown, the average particle size of the obtained A. muciniphila OMVs was 149.9 nm, and they presented a typical bilayer membrane nanostructure under the electron microscope, which was consistent with the A. muciniphila OMVs described in previous studies. Subsequently, CAC mice were exposed to chronic stress and then gavaged with A. muciniphila OMVs. The open field results showed that, compared with the Stress+CAC group (4.697±1.125), after the intervention with A. muciniphila OMVs (7.029±1.410), the exploration distance of chronically stressed mice was significantly increased (t = 3.167, P = 0.01)( Figure 5C ). The tail suspension results showed that after treatment with A. muciniphila OMVs, compared with the mice in the Stress+CAC group (184.5±37.16), the immobility time of the mice in the A. muciniphila+Stress+CAC group (100.9±35.80) was significantly reduced (t = 3.969, P = 0.0026)( Figure 5D ). The above results suggest that A. muciniphila OMVs can relieve the anxiety and depressive emotions of stressed colorectal cancer mice.

[0161] 6. Effects of A. muciniphila on body weight and disease activity index in chronically stressed colorectal cancer mice

[0162] During the treatment of colorectal cancer mice with chronic mild unpredictable stress until the end of the experiment. The results of the body weight change rate showed ( Figure 6 ), compared with the CAC+Stress group, the degree of body weight recovery of the mice in the CAC+Stress+A. muciniphila group was significantly increased (P < 0.05). The results of the disease activity index showed that, compared with the CAC+Stress group, the disease activity index of the mice in the CAC+Stress+A. muciniphila group was significantly decreased (P < 0.001). These results indicate that A. muciniphila can relieve the promoting effects of chronic stress on body weight loss and intestinal function damage in colorectal cancer mice.

[0163] 7. Effects of A. muciniphila on tumor burden mediated by chronic stress in colorectal cancer

[0164] After 14 days of gavage with A. muciniphila in chronically stressed colorectal cancer mice, the gross observation results of the colon tissue showed ( Figure 7) Compared with the mice in the CAC+Stress group, the total number, volume of tumors, and the number of tumors with a size greater than 4 mm in the CAC+Stress+A.muciniphila group of mice were significantly reduced (P < 0.05, P < 0.001, P < 0.05). These results indicate that A.muciniphila can alleviate the promoting effect of chronic stress on the tumor burden in colorectal cancer mice.

[0165] 8. Effects of A.muciniphila on chronic stress-mediated proliferation of colorectal cancer tumor cells

[0166] After 14 days of intragastric administration of A.muciniphila to colorectal cancer mice with chronic stress, the results of immunohistochemistry of tissue sections showed ( Figure 8 ) that compared with the CAC+Stress group, the expression level of Ki67 in the tumor tissue of the CAC+Stress+A.muciniphila group was significantly reduced (P < 0.05). This result indicates that A.muciniphila can alleviate the promoting effect of chronic stress on the cell proliferation activity in the tumor tissue of colorectal cancer mice.

[0167] 9. Effects of A.muciniphila on chronic stress-mediated pathological progression of colorectal cancer tumor tissue

[0168] After 14 days of intragastric administration of A.muciniphila to colorectal cancer mice with chronic stress, the results of HE of tissue sections showed ( Figure 9 ) that compared with the CAC+Stress group, the histopathological score of the tumor tissue in the CAC+Stress+A.muciniphila group was significantly reduced (P < 0.01). This result indicates that A.muciniphila can alleviate the promoting effect of chronic stress on the pathological progression of colorectal cancer tumor tissue.

[0169] 10. Effects of A.muciniphila OMV on body weight and disease activity index in colorectal cancer mice with chronic stress

[0170] During the period when colorectal cancer mice were treated with chronic mild unpredictable stress until the end of the experiment. The results of the body weight change rate showed ( Figure 10) Compared with the CAC+Stress group, the degree of body weight recovery in mice of the CAC+Stress+A.muciniphila OMV group was significantly increased (P<0.05). The results of the disease activity index showed that compared with the CAC+Stress group, the disease activity index of mice in the CAC+Stress+A.muciniphila OMV group was significantly decreased (P<0.05). These results indicate that A.muciniphila OMV can alleviate the promoting effect of chronic stress on body weight loss and intestinal function damage in colorectal cancer mice.

[0171] 11. Effect of A.muciniphila OMV on chronic stress-mediated tumor burden in colorectal cancer

[0172] After 14 days of intragastric administration of A.muciniphila OMV to chronic stress colorectal cancer mice, gross observation of colon tissues showed ( Figure 11 ) that compared with mice in the CAC+Stress group, the number, volume of tumors, and the number of tumors with a size of 2-4 mm in mice of the CAC+Stress+A.muciniphila OMV group were all significantly reduced (P<0.05, P<0.05, P<0.05). These results indicate that A.muciniphila OMV can alleviate the promoting effect of chronic stress on tumor burden in colorectal cancer mice.

[0173] 12. Effect of A.muciniphila OMV on chronic stress-mediated tumor cell proliferation in colorectal cancer

[0174] After 14 days of intragastric administration of A.muciniphila OMV to chronic stress colorectal cancer mice, immunohistochemical results of tissue sections showed ( Figure 12 ) that compared with the CAC+Stress group, the expression level of Ki67 in tumor tissues of the CAC+Stress+A.muciniphila OMV group was significantly reduced (P<0.01). This result indicates that A.muciniphila OMV can alleviate the promoting effect of chronic stress on cell proliferation activity in tumor tissues of colorectal cancer mice.

[0175] 13. Effect of A.muciniphila OMV on chronic stress-mediated pathological progression of colorectal cancer tumor tissues

[0176] After 14 days of intragastric administration of A.muciniphila OMV to chronic stress colorectal cancer mice, HE results of tissue sections showed ( Figure 13) Compared with the CAC+Stress group, the histopathological score of tumor tissues in the CAC+Stress+A.muciniphila OMV group was significantly decreased (P<0.001). This result indicates that A.muciniphila OMV can alleviate the promoting effect of chronic stress on the pathological progression of colorectal cancer tumor tissues.

[0177] It should be noted that although the technical solutions of the present invention are introduced by specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0178] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to technologies in the market, or to enable other ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. Use of Akkermansia muciniphila and / or its outer membrane vesicles in the preparation of a product for relieving and / or inhibiting the progression of chronic stress-mediated colorectal cancer.

2. The use according to claim 1, characterized in that, The relief and / or inhibition of the progression of chronic stress-mediated colorectal cancer includes at least one of (a) to (e): (a) Relieving and / or inhibiting the promoting effect of chronic stress on body weight loss in an organism suffering from colorectal cancer; (b) Relieving and / or inhibiting the promoting effect of chronic stress on intestinal function damage in an organism suffering from colorectal cancer; (c) Relieving and / or inhibiting the promoting effect of chronic stress on tumor burden in an organism suffering from colorectal cancer; (d) Relieving and / or inhibiting the promoting effect of chronic stress on the proliferation activity of colorectal cancer tumor cells; (e) Relieving and / or inhibiting the promoting effect of chronic stress on the pathological progression of colorectal cancer tumor tissue.

3. The use according to claim 2, characterized in that, The tumor burden includes the total number of tumors, tumor volume, and / or the number of tumors larger than 4 mm.

4. The use according to claim 2 or 3, characterized in that, The pathological progression of the tumor tissue includes the growth, invasion, and metastasis of the tumor.

5. Use according to any one of claims 2 to 4, characterized in that The organism includes mammals; Optionally, the mammals include mice, rats, guinea pigs, cows, sheep, cats, dogs, horses, rabbits, pigs, monkeys, and humans.

6. The use according to any one of claims 1 to 5, characterized in that, The product contains one or more of live cells, cell cultures, and cell culture supernatants of Akkermansia muciniphila.

7. The use according to any one of claims 1 to 6, characterized in that, The product includes a drug and / or a pharmaceutical composition.

8. The use according to claim 7, characterized in that, The pharmaceutical composition further contains (i) and / or (ii): (i) One or more pharmaceutically acceptable carriers; (ii) One or more drugs for clinically treating colorectal cancer.

9. The use according to claim 8, characterized in that, The pharmaceutically acceptable carrier includes one or a combination of two or more of a solvent, solubilizer, cosolvent, emulsifier, flavoring agent, odorant, coloring agent, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, pH regulator, stabilizer, surfactant, and preservative.

10. The use according to any one of claims 7 to 8, characterized in that, The pharmaceutical composition is formulated into a solid preparation, semi-solid preparation, or liquid preparation.

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