Application of intestinal symbiotic bacteria in prevention and treatment of colorectal cancer

Abstract

The invention discloses an application of intestinal symbiotic bacteria in preparation of products for preventing and treating colorectal cancer. The intestinal symbiotic bacteria are ruminococcus flavus or succinic acid producing filamentous bacteria. The method has the beneficial effects that the intestinal flora balance can be regulated; the intestinal barrier function can be enhanced; the immunoregulation effect is achieved; the effect of comprehensively preventing and treating colorectal cancer is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of intestinal commensal bacteria in preventing and treating colorectal cancer. Background Art

[0002] Colorectal cancer is a major disease that seriously threatens human health worldwide. Its incidence rate has remained high among all types of malignant tumors, and has shown an increasing trend year by year in many regions. According to relevant data from the World Health Organization, there are a large number of new cases and deaths each year, which has brought heavy medical burdens and mental pressure to society and families.

[0003] Traditional treatments for colorectal cancer mainly include surgical removal of tumor tissue, chemotherapy, and radiotherapy. Although surgical treatment can directly remove the lesion in the early stage, it is often difficult to completely eliminate micro-metastases for patients in the middle and late stages. In addition, the surgery is traumatic and the recovery period is long. Patients face many risks of complications and their quality of life will be greatly affected. Chemotherapy uses various chemical drugs to inhibit the growth and division of tumor cells. However, while these drugs kill tumor cells, they will inevitably damage normal tissue cells, causing a series of serious side effects such as nausea, vomiting, hair loss, and bone marrow suppression. Many patients are forced to interrupt treatment because they cannot tolerate it. Radiotherapy uses radiation to destroy the DNA structure of tumor cells to achieve the purpose of controlling tumor growth, but normal tissues in the radiotherapy area will also be affected by radiation, causing adverse reactions such as radiation inflammation and intestinal dysfunction. In the long run, it may also induce secondary tumors.

[0004] In recent years, the relationship between intestinal microbiota and human health and disease has become a hot topic in the field of medical research. The human intestine is home to a large number of diverse microorganisms, which have formed a complex and close symbiotic relationship with the host during the long process of evolution. As a key component of the intestinal microbiota, intestinal commensal bacteria play an indispensable role in maintaining the normal physiological function of the intestine. For example, they participate in the digestion of food and the absorption of nutrients. They can break down some complex carbohydrates and proteins that are difficult to be directly used by the human body into small molecules, providing the human body with an additional source of energy; they are also involved in the synthesis of certain vitamins and short-chain fatty acids and other metabolites that are beneficial to the human body. These substances are important for maintaining the health of intestinal epithelial cells, regulating intestinal peristalsis, and enhancing the intestinal immune barrier function.

[0005] More and more studies have shown that the imbalance of intestinal microbiota is closely related to the occurrence and development of colorectal cancer. In the intestinal microecological environment of patients with colorectal cancer, significant changes in the composition and diversity of intestinal flora are often observed. Some harmful bacteria that were originally small in number proliferate in large numbers, and they may promote the occurrence of colorectal cancer through various mechanisms. Fusobacterium nucleatum can adhere to the surface of intestinal epithelial cells and invade the cells, triggering local inflammatory responses and activating carcinogenic signaling pathways. At the same time, it can interact with other bacteria to jointly create a microenvironment that is conducive to tumor growth. The number of some beneficial bacteria is relatively reduced, and their protective effect on the intestine is weakened. This imbalance of intestinal flora can lead to impaired intestinal barrier function and increased intestinal permeability, making it easier for bacteria and their toxins, carcinogens, etc. to enter the intestinal submucosal layer, thereby activating the intestinal immune system and causing chronic inflammation. Long-term chronic inflammatory environment can cause a series of pathological changes in intestinal epithelial cells, such as gene mutations, abnormal proliferation, and inhibition of cell apoptosis, which ultimately lead to the formation of colorectal cancer.

[0006] Based on the above-mentioned understanding of the limitations of traditional treatment methods and the in-depth understanding of the important role of intestinal microbiota in the pathogenesis of colorectal cancer, intestinal commensal bacteria have gradually attracted widespread attention as a potential new prevention and treatment target. By regulating the composition and function of intestinal commensal bacteria, it is expected to restore the balance of intestinal microecology, intervene in the occurrence and development of colorectal cancer from the source, and open up new ways for the prevention and treatment of colorectal cancer. This has also become one of the most promising and challenging research directions in the current biomedical field. Summary of the invention

[0007] In order to solve the above problems, especially to address the deficiencies in the prior art, the present invention provides an application of intestinal commensal bacteria in the prevention and treatment of colorectal cancer that can solve the above problems.

[0008] To achieve the above purpose, the present invention adopts the following technical means:

[0009] The invention discloses an application of intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer. The intestinal commensal bacteria are Ruminococcus xanthus or Succinic Acid-producing Filamentobacillus.

[0010] A further embodiment of the present invention is that the Ruminococcus xanthus or Bacillus succinate-producing prevents and treats colorectal cancer by regulating the structure of intestinal flora, improving intestinal barrier function and / or immune regulation.

[0011] A further embodiment of the present invention is that the product for preventing and treating colorectal cancer is an oral preparation, a suppository or an enema.

[0012] A further embodiment of the present invention is that the oral preparation is a capsule, a tablet or a powder.

[0013] A further embodiment of the present invention is that when preparing the oral preparation, the bacteria of Ruminococcus flavus or Bacillus succinici can be mixed with a pharmaceutically acceptable carrier or auxiliary material.

[0014] A further embodiment of the present invention is that the application also includes application in the treatment of colorectal cancer, by combining conventional treatment methods to regulate intestinal microecology, enhance intestinal barrier function and improve intestinal immune status, thereby improving the treatment effect of colorectal cancer and reducing the recurrence rate.

[0015] Beneficial effects of the present invention:

[0016] 1. The present invention can regulate the balance of intestinal flora. Yellow Ruminococcus and Succinic Acid Bacillus can actively participate in the regulation of intestinal flora. Yellow Ruminococcus can effectively inhibit the growth and reproduction of harmful bacteria related to colorectal cancer by secreting special metabolites or competing for nutrients. For example, some antimicrobial peptides it produces can destroy the cell wall or cell membrane structure of harmful bacteria, thereby reducing their colonization number in the intestine. Succinic Acid Bacillus can promote the growth and colonization of beneficial bacteria. It can decompose complex carbohydrates to produce substances such as organic acids, create a more suitable living environment for beneficial bacteria, and increase the number of beneficial bacteria. This optimization and adjustment of the intestinal flora structure can significantly reduce the chronic inflammatory response caused by harmful bacteria in the intestine, reduce the level of inflammatory factors, and thus reduce the risk of colorectal cancer caused by long-term intestinal inflammation, and restore the healthy balance of intestinal microecology.

[0017] 2. The present invention can enhance the intestinal barrier function. The short-chain fatty acids produced by yellow Ruminococcus during intestinal metabolism play a vital role in the intestinal barrier function. Butyric acid can stimulate the proliferation and differentiation of intestinal epithelial cells and increase the number and integrity of intestinal epithelial cells. At the same time, it can also upregulate the expression of tight junction proteins between intestinal epithelial cells, making the connection between intestinal epithelial cells tighter, and effectively preventing harmful substances and bacteria in the intestine from shifting into the intestinal submucosal layer. Succinic acid-producing filamentous bacteria can regulate the secretion and composition of the intestinal mucus layer, making the mucus layer thicker and more viscous, forming a physical barrier to prevent harmful substances from directly contacting intestinal epithelial cells, and helping to maintain the reasonable distribution and functional stability of immune cells in the intestine, jointly enhancing the intestinal barrier function from many aspects, reducing the damage of intestinal epithelial cells to carcinogenic factors, and reducing the incidence of colorectal cancer.

[0018] 3. The present invention has an immunomodulatory effect. Ruminococcus flavus can activate a variety of immune cells in the intestinal immune system, such as macrophages, dendritic cells, etc. It can induce macrophages to polarize to the M1 type, enhance the phagocytic ability and antigen presentation ability of macrophages, and enable them to more effectively identify and eliminate tumor cells and foreign pathogens. At the same time, Ruminococcus flavus can also regulate the secretion of immune factors, promote the production of anti-inflammatory factors, inhibit excessive inflammatory reactions, and avoid intestinal tissue damage and carcinogenesis caused by chronic inflammation. Succinic acid-producing filamentous bacteria participate in the development and maturation of intestinal-associated lymphoid tissue, regulate the secretion of immunoglobulins, enhance the immune defense ability of the intestinal mucosa, enable the intestinal immune system to detect and respond to abnormal changes in tumor cells in a timely manner, play an important role in immune surveillance and regulation in the prevention and treatment of colorectal cancer, and improve the body's overall anti-tumor immunity.

[0019] 4. The present invention has a comprehensive effect of preventing and treating colorectal cancer. The application of yellow Ruminococcus or succinic acid-producing filamentous bacteria to the prevention and treatment of colorectal cancer has significant advantages both in disease prevention and in adjuvant therapy. At the prevention level, by maintaining the balance of intestinal microecology for a long time and enhancing the intestinal barrier and immune function, the risk of colorectal cancer in healthy people can be effectively reduced. In terms of treatment, when combined with traditional treatment methods, the side effects of traditional treatments can be reduced, such as relieving adverse reactions such as intestinal mucosal damage and diarrhea caused by chemotherapy, and improving patients' tolerance to treatment. Moreover, these two bacteria can coordinate with traditional treatment methods from the perspective of intestinal microenvironment regulation, enhance the inhibition and killing effects on tumor cells, improve the effectiveness of treatment, prolong the survival of patients, and improve the quality of life of patients, providing a new and potential comprehensive strategy for the prevention and treatment of colorectal cancer. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1

[0022] The invention discloses an application of intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer. The intestinal commensal bacteria are yellow Ruminococcus or succinic acid-producing filamentous bacteria.

[0023] Ruminococcus xanthus or succinic acid-producing filamentous bacteria can prevent and treat colorectal cancer by regulating the structure of intestinal flora, improving intestinal barrier function and / or immune regulation;

[0024] Regulating the structure of intestinal flora: Ruminococcus flavus can produce some antibacterial substances or inhibit the growth and reproduction of harmful bacteria related to colorectal cancer (such as Fusobacterium nucleatum and certain toxin-producing Escherichia coli) by competing for nutrients, thereby adjusting the composition and proportion of intestinal flora, reducing the intestinal inflammatory response caused by harmful bacteria, and reducing the risk of inflammation-related tumors. Succinic acid-producing filamentous bacteria can also work synergistically with other beneficial bacteria through similar mechanisms to promote the recovery of intestinal flora to a healthy state. For example, it can provide substrates or suitable microenvironments for the growth of beneficial bacteria such as Bifidobacterium, and jointly build a microbial community structure that is beneficial to intestinal health.

[0025] Improve intestinal barrier function: Short-chain fatty acids (such as butyrate) produced by yellow Ruminococcus during fermentation can promote the proliferation and differentiation of intestinal epithelial cells and enhance the expression of tight junction proteins (such as occludin, claudin, etc.) between intestinal epithelial cells, thereby strengthening the intestinal barrier, preventing harmful substances and bacteria from shifting into the intestinal submucosal layer, and blocking the path of tumor occurrence. Succinic acid-producing filamentous bacteria can regulate the secretion and composition of the intestinal mucus layer, increase the barrier function of the mucus layer, protect the intestinal epithelium from invasion by harmful substances and bacteria, and also help maintain immune homeostasis in the intestine, indirectly reducing the incidence of colorectal cancer.

[0026] Immunomodulatory effect: Ruminococcus flavus can stimulate the activity of intestinal immune cells (such as macrophages, dendritic cells, etc.), prompting them to secrete appropriate amounts of immunomodulatory factors (such as interleukin-10, tumor necrosis factor-α, etc.), enhance intestinal immune surveillance, promptly remove abnormal cells that may become cancerous, and play a balancing role in the inflammatory response to avoid the tumor-promoting effect caused by excessive inflammation. Succinic acid-producing filamentous bacteria also participate in the intestinal immune regulation process. It can induce the development and maturation of intestinal-related lymphoid tissue, regulate the secretion of immunoglobulins, and keep the intestinal immune system in a suitable activation state, effectively resisting the occurrence and development of tumors.

[0027] Yellow Ruminococcus or succinic acid-producing filamentous bacteria can be made into various forms of products for preventing and treating colorectal cancer. For example, oral preparations include capsules, tablets, powders, etc. When preparing oral preparations, the bacteria of yellow Ruminococcus or succinic acid-producing filamentous bacteria can be mixed with pharmaceutically acceptable carriers or excipients. Commonly used carriers or excipients include starch, lactose, sucrose, mannitol, microcrystalline cellulose, sodium carboxymethyl cellulose, magnesium stearate, etc. For capsules, the cultured bacteria can be centrifuged, washed, freeze-dried, etc. to obtain bacterial powder, which is filled into the capsule shell according to a certain dose; tablets are made by mixing the bacterial powder and excipients evenly and then using a tablet press; powders are made by directly drying and crushing the bacteria. It can also be made into intestinal local administration preparations such as suppositories or enemas to act more directly on the intestinal lesions. Suppositories can be made by using cocoa butter, semi-synthetic fatty acid glycerides, etc. as a matrix, in which the bacteria are evenly dispersed; enemas can be made by mixing the bacteria with suitable buffer solutions, preservatives, etc. to form solution-type, suspension-type, or emulsion-type enema solutions.

[0028] Applications also include use in the treatment of colorectal cancer. By combining conventional treatments, it can regulate the intestinal microecology, enhance intestinal barrier function, and improve intestinal immune status, thereby improving the treatment effect of colorectal cancer and reducing the recurrence rate.

[0029] Yellow Ruminococcus or succinic acid-producing filamentous bacillus can be combined with other ingredients with synergistic prevention and treatment of colorectal cancer to form a pharmaceutical composition. For example, it can be used in combination with natural plant extracts (such as curcumin, resveratrol, etc.), which have antioxidant, anti-inflammatory, anti-tumor and other activities. When acting together with yellow Ruminococcus or succinic acid-producing filamentous bacillus, the occurrence and development of colorectal cancer can be inhibited from different angles. For example, curcumin can regulate the intracellular signaling pathway to enhance the inhibition of tumor cells, and yellow Ruminococcus regulates intestinal flora and immunity to further synergize. It can also be combined with chemotherapeutic drugs (such as 5-fluorouracil, oxaliplatin, etc.) to improve the therapeutic effect of tumors while reducing the side effects of chemotherapeutic drugs. Chemotherapy drugs directly kill tumor cells, and intestinal commensal bacteria improve the intestinal environment to promote the action of chemotherapeutic drugs and reduce their damage to the intestine. It can also be combined with immunotherapy drugs (such as anti-PD-1 antibodies, anti-CTLA-4 antibodies, etc.). Intestinal commensal bacteria regulate the intestinal immune microenvironment so that immunotherapy drugs can better activate the immune system to kill tumor cells. The two synergistically enhance the efficacy of immunotherapy.

[0030] Example 2

[0031] Isolation and culture of Ruminococcus xanthus and Succinimidobacterium succinigensis.

[0032] Fresh stool samples from healthy volunteers were collected, stored in an anaerobic environment and processed in time. After gradient dilution with sterile anaerobic buffer, the stool samples were inoculated on selective culture plates specifically used for culturing yellow Ruminococcus and succinic acid-producing Bacillus. The selective culture medium for yellow Ruminococcus may contain specific cellulose substances as carbon sources and appropriate amounts of rumen fluid extracts and other components. The culture medium for succinic acid-producing Bacillus may add corresponding sugars, amino acids and other components according to its nutritional needs, and add appropriate amounts of antibiotics to inhibit the growth of miscellaneous bacteria. The inoculated plates were placed in an anaerobic incubator and cultured at 37°C for 48-72 hours. The morphology of the colonies growing on the plates was observed, and the colonies suspected of yellow Ruminococcus and succinic acid-producing Bacillus were selected. Pure colonies were obtained by multiple streaking purification and culture. Gram staining, physiological and biochemical identification, and 16SrRNA gene sequencing analysis were performed on the pure colonies to determine whether they were yellow Ruminococcus or succinic acid-producing Bacillus.

[0033] Example 3

[0034] Preventive effects of yellow Ruminococcus and succinic acid-producing Bacillus on colorectal cancer mouse model. A certain number of C57BL / 6 mice were randomly divided into three groups: control group, yellow Ruminococcus treatment group and succinic acid-producing Bacillus treatment group. The mice in the yellow Ruminococcus treatment group and succinic acid-producing Bacillus treatment group were given the corresponding bacterial solution by gavage, with a bacterial solution concentration of 1×10^8-1×10^10 CFU / ml and a gavage volume of 0.2-0.5ml, 3-5 times a week, for 4-8 weeks. The mice in the control group were given an equal amount of sterile buffer. After the gavage, the colorectal cancer mouse model was established by AOM / DSS induction method. During the modeling process, the weight changes, fecal characteristics and other indicators of the mice were continuously observed. After the experiment, the mice were dissected to observe the occurrence of colon tumors, count the number and size of tumors, and perform pathological examinations on colon tissues. The results showed that the tumor incidence, number and size of mice in the groups treated with Ruminococcus flavus and Bacillus succinigensis were significantly lower than those in the control group, indicating that Ruminococcus flavus and Bacillus succinigensis have a good effect in preventing colorectal cancer.

[0035] Example 4

[0036] Effects of yellow Ruminococcus and succinic acid-producing Bacillus on the proliferation and apoptosis of colorectal cancer cells. Human colorectal cancer cell lines (such as SW480, HT-29, etc.) were inoculated in 96-well plates or culture dishes. After culturing to the logarithmic growth phase, different concentrations of yellow Ruminococcus and succinic acid-producing Bacillus cell lysates or culture supernatants were added, and the culture was continued for a certain period of time. The MTT method was used to detect cell proliferation activity, and the Annexin V-FITC / PI double staining method was used to detect cell apoptosis rate. The results showed that yellow Ruminococcus and succinic acid-producing Bacillus could significantly inhibit the proliferation of colorectal cancer cells and induce their apoptosis, and it was concentration-dependent.

[0037] The examples given in the present invention are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here, and the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer, characterized in that: The intestinal commensal bacteria are yellow Ruminococcus or succinic acid-producing filamentous bacteria.

2. The use of an intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer according to claim 1, characterized in that: The yellow Ruminococcus or succinic acid-producing filamentous bacteria prevent and treat colorectal cancer by regulating the intestinal flora structure, improving the intestinal barrier function and / or immune regulation.

3. The use of an intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer according to claim 2, characterized in that: The colorectal cancer prevention and treatment product is an oral preparation, a suppository or an enema.

4. The use of an intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer according to claim 3, characterized in that: The oral preparation is a capsule, a tablet or a powder.

5. The use of an intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer according to claim 4, characterized in that: When preparing the oral preparation, the bacteria of Ruminococcus xanthus or Bacillus succinici can be mixed with pharmaceutically acceptable carriers or excipients.

6. The use of an intestinal commensal bacteria in the preparation of a product for preventing and treating colorectal cancer according to claim 1, characterized in that: The application also includes application in the treatment of colorectal cancer, which improves the treatment effect of colorectal cancer and reduces the recurrence rate by combining conventional treatment methods to regulate intestinal microecology, enhance intestinal barrier function and improve intestinal immune status.