Application of bacteroides ovale in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention relates to the technical field of bacteroides application, and discloses a bacteroides ovatus strain capable of improving or treating hepatocellular carcinoma, the name of the bacteroides ovatus strain is BAA-1304, the bacteroides ovatus strain is colonized in the intestinal tract of a mouse in an intragastric administration mode, subcutaneous isotransplantation tumors of a hepatocellular carcinoma cell line Hepa1-6 can be obviously reduced, and the hepatocellular carcinoma can be improved or treated. The effects of improving and treating the liver cancer are exerted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of Bacteroides ovatus in preparation of a hepatocellular carcinoma treatment drug. BACKGROUND

[0002] Liver cancer is the sixth most common cancer in the world, ranking third in the most common causes of cancer-related deaths, and the number of new and death cases is huge every year. Hepatocellular carcinoma (HCC) is the most common primary liver cancer, accounting for more than 80%, and the risk factors for the disease include viral hepatitis, alcohol-related liver disease and metabolic dysfunction-related fatty liver disease, etc. Epidemiological surveys have found that about 85% of cancers in China are related to hepatitis B virus (HBV), and about 40% of liver cancers occur in China, and more than 70% of cases are found in the late stage. Liver cancer is highly malignant, progresses rapidly, and has a poor prognosis, which seriously threatens human life and health, and also brings a heavy medical burden.

[0003] In the past two decades, immune checkpoint inhibitors (ICIs) treatment programs have been used as a first-line guideline in clinical practice, and the prognosis of patients with advanced hepatocellular carcinoma has been improved to a certain extent, but in clinical practice, the efficiency of programmed cell death-1 (PD-1) and its ligand (PD-L1) inhibitors is about 10%-20%, and in the second-line treatment, it is less than 10%, and as many as 30%-40% of patients do not respond to these drugs. At the same time, further breakthroughs are still needed for the treatment of early HCC, and among the diagnosed cases of liver cancer, about 30% of patients are diagnosed with early HCC, and the current treatment of early HCC mainly includes surgical methods such as tumor resection, liver transplantation or local ablation, but as many as 30%-50% of patients will have disease recurrence within 3 years, and 70% of patients will have recurrence within 5 years after resection or local ablation. Therefore, exploring new liver cancer treatment or adjuvant treatment methods provides a new anticancer strategy for liver cancer patients, which has important clinical significance and application value.

[0004] Studies have found that the gut microbiota plays a crucial role in the progression of chronic liver disease and the development of liver cancer, and also plays a key role in the anti-tumor response after immunotherapy and chemotherapy by regulating immune responses. Gut microbiota dysbiosis is common in liver cancer patients, and many studies have begun to examine changes in the gut microbiota of liver cancer or cirrhosis patients to explore its potential as a non-invasive biomarker or adjuvant therapy for liver cancer. The gut microbiota may affect liver cancer progression through multiple mechanisms, such as producing metabolites with carcinogenic or anti-tumor activities and influencing the host's immune response. Currently, there is a lack of conclusive evidence to support that a single gut microbiota can effectively inhibit liver cancer progression, and further research is needed into the mechanisms by which the gut microbiota regulates liver cancer.

[0005] Our previous research group used third-generation sequencing of the gut microbiota in clinical liver cancer samples and found that *Bacteroides ovalis* was abundant in the gut of healthy individuals, while its abundance was reduced in the gut of liver cancer patients, suggesting that it may play a role in inhibiting disease progression. Bacteroidetes are one of the major components of the gut microbiota, accounting for over 90% of the total gut bacteria along with Firmicutes. Due to the large number and diverse functions of Bacteroidetes, the specific roles of individual species in disease are still not fully understood. Summary of the Invention

[0006] The purpose of this invention is to explore and verify the phenotype and mechanism of Bacteroides ovale in inhibiting liver cancer, and to propose its application in the preparation of drugs for the prevention and treatment of liver cancer. This objective is achieved through the following technical solutions: Bacteroides ovalis ( Bacteroidetes. ovatus Its application in the preparation of drugs for the treatment and prevention of hepatocellular carcinoma.

[0007] Furthermore, the hepatocellular carcinoma prevention drug is an oral preparation containing live Bacteroides ovalis; the dosage form is granules, capsules, tablets, powders, oral liquids, suspensions, or emulsions. Preferably, it is an oral live bacterial liquid.

[0008] Furthermore, the hepatocellular carcinoma treatment drug is an oral preparation containing live Bacteroides ovalis. Preferably, it is an oral live bacterial solution.

[0009] The advantages and beneficial effects of this invention are: This invention, through in-depth research, reveals and verifies for the first time the key intestinal symbiotic bacterium—Bacteroides ovalis (… Bacteroidetes. ovatusThe core regulatory role and mechanism of this strain in hepatocellular carcinoma immunotherapy. This strain was found to significantly inhibit the occurrence and development of hepatocellular carcinoma. Its mechanism of action lies in inhibiting tumor proliferation, promoting tumor apoptosis, reshaping the tumor immune microenvironment, effectively relieving immunosuppression, and strongly activating the host's anti-tumor immune response. Based on this original scientific discovery, this invention pioneered the development of a live Bacteroides ovalis preparation for the application in the preparation of drugs for the prevention and treatment of liver cancer. This provides a novel, microbiome-based biotherapy strategy for overcoming the clinical challenge of drug resistance in liver cancer immunotherapy. Attached Figure Description

[0011] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0012] Figure 1 The results of the experiment show the effect of early colonization of B. ovatus on allogeneic tumors of the Hepa1-6 liver cancer cell line; Early colonization of Bacteroides ovale inhibits the growth of hepatocellular carcinoma. Figure 1 A schematic diagram of the mouse treatment process. Figure 1 Typical tumor images of mice in the control group and the early treatment group with Bacteroides ovalis. Figure 1 C and Figure 1 D represents the tumor change curves over time and the tumor weight statistics after tumor removal in mice in the control group and the early treatment group of Bacteroides ovalis, respectively.

[0013] Figure 2 The results of an experiment on the effects of late-stage complementation of B. ovatus on allogeneic hepatocellular carcinoma lines Hepa1-6; Late-stage colonization of Bacteroides ovale inhibits the growth of hepatocellular carcinoma. Figure 2 A schematic diagram of the mouse treatment process. Figure 2 Typical tumor images of mice in the control group and the late-stage treatment group with Bacteroides ovalis. Figure 2 C and Figure 2 D represents the tumor growth curves over time in the control group and the late-stage Bacteroides ovale treatment group (C), and the tumor weight statistics after tumor removal (D), respectively. The arrows indicate the time when bacterial gavage treatment began.

[0014] Figure 3 For early planting B. ovatus The impact on T cells in the tumor microenvironment.

[0015] Early colonization of Bacteroides ovale increases CD8 levels in the tumor microenvironment. + The proportion of T cells. Figure 3 A represents the detection of CD8 in mouse tumors by flow cytometry. + T and CD4 + A proportional diagram of T. Figure 3B represents the detection of CD8 in mouse tumors by flow cytometry. + T and CD4 + A statistical chart of the proportions of T.

[0016] Figure 4 For early planting B. ovatus Effects on tumor cell proliferation and apoptosis;

[0017] Figure 4 A is an immunohistochemical Ki67 staining image. Figure 4 B is the immunohistochemical Ki67 statistical plot. Figure 4 C is the apoptosis tunnel staining image. Figure 4 D is the apoptosis tunnel statistics graph. Detailed Implementation

[0019] Bacteroides ovalis ( Bacteroidetes. ovatus In this embodiment, Bacteroides ovalis is deposited in the American Type Culture Collection (ATCC) with accession number BAA-1304 in the preparation of drugs for the treatment and prevention of hepatocellular carcinoma.

[0020] Experimental Example 1 Culture of Bacteroides ovalis 1. Bacteroides ovatus (ATCC BAA-1304), purchased from the American Type Culture Collection (ATCC). 2. The purchased strain was aseptically inoculated into liquid culture medium and then cultured in an anaerobic workstation containing 5% carbon dioxide, 10% hydrogen and 85% nitrogen.

[0021] 3. The culture medium formulation is shown in Table 1.

[0022] Table 1 Experimental Example 2 Early planting B. ovatus Effects on Hepa1-6 hepatocellular carcinoma cell line allografts Six 25-day-old C57BL-6J mice (sex or no sex) were selected and treated with a mixture of antibiotics in water for 7 days. The specific antibiotic composition is shown in Table 2, which was used to disrupt the intestinal microbiota of the experimental mice.

[0023] Table 2 The mice were then randomly divided into two groups: the control group was given bacterial culture medium by gavage, and the treatment group was given bacterial culture medium by gavage. B. ovatus Active bacterial solution (OD) 600 =1, ≈1×10 8 CFU / ml, converted to human dosage, is approximately 8 × 10⁻⁶. 6 Administer 200 μL / animal (cfu / kg) three times a week for one week. Then, cultured Hepa1-6 cell lines were injected at a rate of 1 × 10⁻⁶. 6 The bacteria were injected subcutaneously into the left groin of mice, marked as day 0. Bacterial colonization was then maintained three times per week, with tumor volume measured every other day. Tumor growth curves were plotted for both groups of mice. On day 22, the subcutaneous tumors were removed, and the size and weight of the tumors in both groups were recorded by photograph. It was found that... B. ovatus After treatment, tumor growth in tumor-bearing mice was significantly inhibited. From day 18 post-inoculation, a statistically significant difference in tumor volume was observed between the treatment group and the control group (p<0.05), and this difference persisted until the end of the experiment. Ultimately, B. ovatus The tumor weight in the treated mice was significantly lower than that in the control group (p<0.001). 。 Explanation of early replenishment B. ovatus It can inhibit tumor growth. Specific results are as follows: Figure 1 As shown.

[0024] Experimental Example 3 Late replenishment Five 25-day-old C57BL-6J mice (sex not limited) were selected and treated with a mixture of water and antibiotics for 7 days. The mice were then randomly divided into two groups, and the cultured Hepa1-6 cell line was administered at a rate of 1×10⁻⁶. 6 The bacteria were injected subcutaneously into the left groin of mice, marked as day 0. Tumor volume was measured every other day, and tumor growth curves were plotted for both groups. On day 7, mice were divided into two groups based on tumor volume, ensuring that the average tumor size of the control and treatment groups was similar. The control group was supplemented with bacterial culture medium by gavage, while the treatment group was supplemented with... B. ovatus Active bacterial solution (OD) 600 =1, ≈1×10 8 (CFU / ml), 200 μL / mouse, three times a week. At day 17, subcutaneous tumors were harvested from the mice, and the size and weight of the tumors in both groups were recorded by photograph. B. ovatus After treatment, tumor growth in tumor-bearing mice was significantly inhibited. From day 15 post-inoculation, a statistically significant difference in tumor volume was observed between the treatment group and the control group (p<0.05), and this difference persisted until the end of the experiment. Ultimately, B. ovatus The tumor weight in the treated mice was significantly lower than that in the control group (p<0.001).。 Explanation of later replenishment B. ovatus It can inhibit tumor growth. Specific results are as follows: Figure 2 As shown.

[0025] Test Example 4 Early planting B. ovatus Effects on T cells in the tumor microenvironment Using the same mouse treatment protocol as in Example 2 above, tumors from mice treated for 22 days were collected and isolated into single cells using a tumor single-cell dissociation kit. The cells were then stained sequentially with CD45, CD3, CD4, and CD8, and CD45 was analyzed by flow cytometry. ＋ / CD3 ＋ / CD4 ＋ CD4 + The percentage of T cells and CD45 ＋ / CD3 ＋ / CD8 ＋ CD8 + The proportion of T cells, compared B. ovatus Effects on T cells in the tumor immune microenvironment, such as Figure 3 As shown, it was found B. ovatus Processing and improving CD8 + The proportion of T cells, and the proportion of CD4 + The effect of the T cell ratio was not significant.

[0026] Experimental Example 5 Early planting B. ovatus Effects on tumor cell proliferation and apoptosis Frozen sections of tumor tissue samples from Experiment 2 were prepared for subsequent Ki-67 and Tunnel staining. Slides fixed in 4% paraformaldehyde were rehydrated and blocked in PBS containing 1% BSA. Primary antibody was incubated overnight at 4°C in blocking buffer. For immunohistochemistry, sections were incubated with anti-rabbit ImmPRESS-HRP-conjugated secondary antibody and stained with DAB, counterstained with hematoxylin. For immunofluorescence staining, secondary antibody was applied to PBS for 1 hour at room temperature, and sections were counterstained with DAPI. Results are as follows. Figure 4 As shown in the figure, compared with the control group, the tumor proliferation signal in the treatment group was significantly reduced and the tumor apoptosis signal was significantly increased.

[0027] Example 1: Bacteroides ovalis ( Bacteroidetes. ovatus The application of this drug in the preparation of a hepatocellular carcinoma prevention drug, wherein the hepatocellular carcinoma prevention drug is an oral live bacterial solution containing live Bacteroides ovalis. The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8cfu / ml.

[0028] Example 2: Bacteroides ovalis ( Bacteroidetes. ovatus The application of this drug in the preparation of hepatocellular carcinoma treatment drugs, wherein the hepatocellular carcinoma prevention drug is an oral live bacterial solution containing live Bacteroides ovalis. The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8 cfu / ml.

[0029] Example 3: Bacteroides ovalis ( Bacteroidetes. ovatus The application of this drug in the preparation of a hepatocellular carcinoma prevention drug, wherein the hepatocellular carcinoma prevention drug is a tablet containing live Bacteroides ovalis. The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8 cfu / g.

[0030] Example 4: Bacteroides ovalis ( Bacteroidetes. ovatus The application of this drug in the preparation of hepatocellular carcinoma treatment drugs, wherein the hepatocellular carcinoma prevention drug is a powder containing live Bacteroides ovalis. The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8 cfu / g.

[0031] Example 5: Bacteroides ovalis ( Bacteroidetes. ovatus The application of this drug in the preparation of a hepatocellular carcinoma prevention drug, wherein the hepatocellular carcinoma prevention drug is a capsule containing live Bacteroides ovalis. The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8 cfu / g.

[0032] Example 6: Bacteroides ovalis ( Bacteroidetes. ovatus The application of this drug in the preparation of hepatocellular carcinoma treatment drugs, wherein the hepatocellular carcinoma prevention drug is an emulsion containing live Bacteroides ovalis. The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8 cfu / mL.

[0033] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. Bacteroides ovalis ( Bacteroidetes. ovatus Its application in the preparation of drugs for the treatment and prevention of hepatocellular carcinoma.

2. The application according to claim 1, characterized in that: The hepatocellular carcinoma prevention drug is an oral preparation containing live Bacteroides ovalis; the dosage form is granules, capsules, tablets, powders, oral liquids, suspensions, or emulsions.

3. The application according to claim 2, characterized in that: The hepatocellular carcinoma prevention drug is an oral live bacterial solution containing live Bacteroides ovalis.

4. The application according to claim 3, characterized in that: The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8 cfu / ml.

5. The application according to claim 1, characterized in that: The hepatocellular carcinoma treatment drug is an oral preparation containing live Bacteroides ovalis.

6. The application according to claim 5, characterized in that: The hepatocellular carcinoma treatment drug is an oral live bacterial solution containing live Bacteroides ovalis.

7. The application according to claim 6, characterized in that: The concentration of Bacteroides ovalis in the oral live bacterial solution is 1×10⁻⁶. 7 ~1×10 8 cfu / ml.

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