Ligilactobacillus animalis and use thereof

Abstract

Ligilactobacillus animalis and a use thereof. The Ligilactobacillus animalis is preserved in Guangdong Microbial Culture Collection Center on 13 October 2022, and the preservation number is GDMCC NO. 62867. The Ligilactobacillus animalis HHP002 can effectively inhibit the mRNA expression of proinflammatory cytokines of human colon cancer cells, thereby inhibiting generation of proinflammatory cytokines, and further inhibiting development of tumors, and the Ligilactobacillus animalis HHP002 can be applied to prevention and treatment of human colon cancer.

Description

Animal combined lactobacillus and its application Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to an animal-associated lactobacillus strain and application thereof. Background Art

[0002] There are many microorganisms living in the human intestines. These microorganisms not only participate in the digestion of food, but also affect human health.

[0003] A growing number of studies are now finding that intestinal microbes can even influence the immune system. For example, one study showed that after fecal microbiota transplantation, the number and function of intestinal T cells in septic mice were restored to a certain extent, and the length of the colon was significantly restored. Histopathological results also showed that inflammation and damage in the small intestine and colon were significantly alleviated. During the development of inflammation, cells produce various cytokines, which not only induce the proliferation and aggregation of immune cells but also may promote the development of tumor cells.

[0004] Colorectal cancer is a highly prevalent cancer. Currently, the available treatment options for cancer are relatively limited, primarily consisting of targeted drugs and chemotherapy. Targeted drugs are expensive and only suitable for patients with specific targets, limiting their potential for treatment. While chemotherapy is inexpensive, it carries significant side effects and can severely reduce a person's immune system.

[0005] Gut microbes are the microorganisms that naturally inhabit the human intestine, including a large number of beneficial probiotics. Gut microbes are inexpensive to produce, and some can influence immune regulation. Using gut microbes to treat colorectal cancer has the potential to further improve the quality of treatment and reduce costs, but no relevant reports have yet been published.

[0006] Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an animal-derived Lactobacillus strain, aiming to improve the quality of treatment for intestinal cancer and reduce the cost of treatment.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A strain of animal-associated Lactobacillus, the animal-associated Lactobacillus (Ligilactobacillus animalis) was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 13, 2022, with the deposit number GDMCC NO.62867.

[0010] A use of the above-mentioned animal combined lactobacillus in preparing a drug for inhibiting the expression of pro-inflammatory cytokines in human colon cancer.

[0011] In the application, the pro-inflammatory cytokine is one or more of IL-1β, IL-6, IL-8, and TNF-α.

[0012] A use of the above-mentioned animal combined lactobacillus in preparing a medicine for treating human colon cancer.

[0013] A use of the above-mentioned animal combined lactobacillus in preparing a drug for preventing human colon cancer.

[0014] A use of the above-mentioned animal-combined lactobacillus in the preparation of a probiotic agent. Beneficial effects:

[0015] The present invention provides a strain of animal-associated Lactobacillus, the deposit number of which is GDMCC NO.62867. After acting on human colon cancer cells, the strain can significantly inhibit the upregulation of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IL-8 in human colon cancer cells, and can be used as a drug for preventing and treating human colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG1 is the detection result of IL-1β, IL-6, IL-8, IL-17 and TNF-α gene expression levels of HT29 cells in the cell experiment of Example 1.

[0017] Figure 2 shows the results of the acid resistance experiment of animals combined with Lactobacillus HHP002.

[0018] FIG3 shows the results of the bile salt tolerance experiment of animals combined with Lactobacillus HHP002.

[0019] FIG4 shows the results of the cytotoxicity test of animals combined with Lactobacillus HHP002. DETAILED DESCRIPTION

[0020] The present invention provides an animal-associated lactobacillus and its application. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] In the following experiments, animal-associated Lactobacillus HHP002 is animal-associated Lactobacillus A-11-15 (Ligilactobacillus animalis) deposited in Guangdong Provincial Microbiological Culture Collection Center on October 13, 2022, with the deposit number GDMCC NO.62867.

[0022] Example 1 Cell experiment

[0023] 1. Cultivation of Human Colon Cancer Cells HT29

[0024] Human colon cancer cells HT29 were cultured in RPMI1640 (HyClone, UT, USA) medium containing 10% FBS (Gibco, Grand Island, NE, USA) and 1% antibiotics (100 mg / mL streptomycin and 100 U / mL penicillin) at 37°C in a humidified atmosphere of 5% CO2. HT29 cells were plated at 5 × 10 5 The cells were seeded at a density of 10 cells / well in a 12-well cell culture plate.

[0025] 2. Co-culture of animal-derived Lactobacillus HHP002 (Ligilactobacillus animalis) with HT29 cells

[0026] 2.1 The experiment was divided into four groups: control group, HHP002 group, LPS group, and LPS+HHP002 group.

[0027] 2.2 The specific operation of the control group is as follows: HT29 cells were cultured without other treatments;

[0028] The specific operation of the HHP002 group was as follows: when the HT29 cells reached 80% confluence, the HT29 cells were placed in a medium containing Lactobacillus animalis HHP002 (the concentration of bacteria was 1×10 7 CFU / mL) cell culture medium and incubated for 6 h;

[0029] The specific operation of the LPS group was as follows: when HT29 cells reached 80% confluence, 10 μg / mL LPS was added and treated for 12 h;

[0030] The specific operation of LPS+HHP002 group was as follows: when HT29 cells reached 80% confluence, HT29 cells were first placed in the presence of animal-associated Lactobacillus HHP002 (bacterial concentration was 1×10 7 CFU / mL) of cell culture medium for 6 h, and then treated with 10 μg / mL LPS for 12 h;

[0031] 2.3 After treatment in each group, the cells were washed three times with PBS and then lysed using RNAiso170Plus (Takara, Dalian, China) to extract RNA.

[0032] 3. Measure the expression levels of cytokine genes. The corresponding results are shown in Figure 1:

[0033] In Figure 1, there was no significant difference in the relative mRNA expression of cytokines in HT29 cells between the Control group and the HHP002 group, and the relative expression levels were very close, indicating that the combined action of animal and lactobacillus HHP002 on human colon cancer cells did not cause upregulation of cytokine expression in human colon cancer cells;

[0034] There were significant differences in the relative mRNA expression levels of cytokines in HT29 cells between the Control group and the LPS group, indicating that LPS can cause the upregulation of multiple proinflammatory cytokines in human colon cancer cells.

[0035] Several cytokines shown in Figure 1 are closely related to the occurrence and development of tumors;

[0036] Among them, IL-1β has been shown to promote the growth and transplantation of various tumors and also cause fever and cachexia;

[0037] IL-6 promotes the formation of tumor blood vessels. In addition, IL-6 also suppresses the body's immune function and regulates the expression of cell cycle proteins through STAT3 activation, causing tumor cells to proliferate faster.

[0038] IL-8 enhances the migration and infiltration capabilities of tumor cells and mediates the development of drug resistance in tumor cells through the PI3K / AKT / mTOR pathway. Furthermore, IL-8 inhibits the activity of cytotoxic T cells and induces the transformation of other immune cells into tumor-associated cells. Therefore, in clinical practice, serum levels of IL-8 are often negatively correlated with the therapeutic efficacy of cancer patients.

[0039] IL-17 can act on fibroblasts, endothelial cells, and tumor cells in the tumor microenvironment, inducing them to produce more pro-inflammatory cytokines such as the aforementioned TNF-α, IL-6, and IL-1β. These cytokines can aggravate the inflammatory response and promote the infiltration of more inflammatory cells into tumor tissues.

[0040] TNF-α can induce tumor recurrence and metastasis of tumor cells.

[0041] Comparing the LPS group with the LPS+HHP002 group alone, the relative mRNA expression levels of IL-1β, IL-6, IL-8 and TNF-α in HT29 cells of the LPS+HHP002 group were significantly lower than those in the LPS group, while the relative mRNA expression levels of IL-1β and TNF-α in the LPS+HHP002 group were significantly different from those in the LPS group, indicating that animal combined with Lactobacillus HHP002 can effectively inhibit the expression of proinflammatory cytokines in human colon cancer cells, thereby inhibiting the further development of tumors. When used in combination with other drugs, it is expected to achieve better therapeutic effects.

[0042] In addition, animal-derived Lactobacillus HHP002 can also be used to prepare early preventive drugs, such as preventing the occurrence and recurrence of human colon cancer. The preventive drug can be in the form of a probiotic agent.

[0043] Example 2 Acid and choline resistance experiments

[0044] The pH value of human gastric juice varies between 1.5 and 4.5. If the bacterial strain cannot survive in a low pH environment, it will affect its intake method; the human large intestine contains bile salts, and its concentration is between 0.3% and 0.5%. The presence of bile salts will inhibit the growth of certain microorganisms. Therefore, growth and survival under low pH and bile salts are considered to be the most ideal characteristics of future probiotic strains.

[0045] The specific steps of the acid resistance test are as follows:

[0046] The pH of the culture medium was adjusted to 2.0, 3.0, and 4.0 with 1M HCl, and then inoculated with animal-associated Lactobacillus HHP002 and cultured at 37°C for 12 hours. The OD values ​​at 0 and 12 hours were measured to determine the acid resistance of the culture medium compared with the untreated control group. The corresponding test results are shown in Figure 2.

[0047] As can be seen from Figure 2, animal-associated Lactobacillus HHP002 has good acid resistance and can still grow under low pH conditions.

[0048] The specific steps of the choline tolerance test are as follows:

[0049] First, 0.1%, 0.3% and 0.5% (w / v) taurine were added to the culture medium, and the culture medium was shaken thoroughly to completely dissolve the taurine. Then, animal-associated Lactobacillus HHP002 was inoculated and cultured at 37°C for 12 hours. The OD values ​​at 0 and 12 hours were measured, respectively, compared with the untreated control group, to determine the bile salt tolerance. The corresponding test results are shown in Figure 3;

[0050] As can be seen from FIG3 , 0.1%, 0.3% and 0.5% (w / v) taurine had no effect on the growth of Lactobacillus animalis HHP002, indicating that Lactobacillus animalis HHP002 has the ability to survive in the human intestine.

[0051] Example 4 Lactate dehydrogenase (LDH) cytotoxicity test

[0052] The specific experimental steps are as follows:

[0053] The experimental subjects were human colon cancer cells HT29, and the experiment was divided into five groups:

[0054] Cell culture medium group;

[0055] Control cell group treated at 37°C (Control group): HT29 cells were cultured without other treatments;

[0056] Animal combined Lactobacillus HHP002 group: When HT29 cells reached 80% confluence, HT29 cells were placed in a medium containing animal combined Lactobacillus HHP002 (bacterial concentration was 1×10 7 CFU / mL) cell culture medium and incubated for 6 h;

[0057] LPS group: when HT29 cells reached 80% confluence, 10 μg / mL LPS was added and treated for 12 h;

[0058] LPS+animal combined Lactobacillus HHP002 group: When the HT29 cells reached 80% confluence, they were first placed in a medium containing animal combined Lactobacillus HHP002 (bacterial concentration was 1×10 7 CFU / mL) of cell culture medium for 6 h, and then treated with 10 μg / mL LPS for 12 h;

[0059] After treatment, add LDH detection working solution, mix well, incubate at room temperature (about 25°C) in the dark for 30 min, and then measure the absorbance at 490 nm;

[0060] The calculation formula for cytotoxicity or LDH enzyme activity is:

[0061] Cytotoxicity or LDH enzyme activity = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100.

[0062] FIG4 is the result of the cytotoxicity test. As can be seen from FIG4 , there is no significant difference in the LDH release between the animal combined with Lactobacillus HHP002 group and the control group, indicating that the animal combined with Lactobacillus HHP002 has no toxicity to cells.

[0063] However, there was a significant difference in the amount of LDH released by the LPS group and the Control group. After LPS treatment, the amount of LDH released by HT29 cells was significantly increased, indicating that LPS could damage HT29 cells.

[0064] The LDH release in the LPS + Lactobacillus spp. HHP002 group was significantly lower than that in the LPS group, indicating that pre-incubation of HT29 cells with Lactobacillus spp. HHP002 significantly reduces LPS-induced damage. When damaged, cells release proinflammatory cytokines. For patients recovering from cancer, overexpression of proinflammatory cytokines may cause tumor recurrence. Therefore, Lactobacillus spp. HHP002 can be used as a probiotic or in the preparation of preventive drugs to reduce damage to intestinal cells, thereby reducing inflammation and tumor recurrence.

[0065] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A strain of animal-associated Lactobacillus, characterized in that: The animal-associated Lactobacillus (Ligilactobacillus animalis) was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 13, 2022, with the collection number GDMCC NO.62867.

2. Use of the animal-combined lactobacillus as claimed in claim 1 in the preparation of a drug for inhibiting the expression of pro-inflammatory cytokines in human colon cancer.

3. The use according to claim 2, characterized in that: The pro-inflammatory cytokines are one or more of IL-1β, IL-6, IL-8, and TNF-α.

4. Use of the animal combined lactobacillus as claimed in claim 1 in the preparation of a drug for treating human colon cancer.

5. Use of the animal combined lactobacillus as claimed in claim 1 in the preparation of a drug for preventing human colon cancer.

6. Use of the animal-associated lactobacillus as claimed in claim 1 in the preparation of a probiotic agent.

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