Application of lactobacillus brevis in inhibition of tumor hepatic metastasis
By using Lactobacillus brevis to reduce the proportion of CD11B+F4/80+ macrophages in the tumor microenvironment, the problem of poor effectiveness in inhibiting tumor liver metastasis was solved, and a significant inhibitory effect of tumor liver metastasis was achieved.
Patent Information
- Application Number
- CN202411482117.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2025-03-04
AI Technical Summary
The prior art is not effective in inhibiting tumor liver metastasis, especially in the immune tolerance environment of the liver, where tumor cells are difficult to be effectively inhibited.
Lactobacillus brevis is used as the active ingredient of the drug, and the proportion of CD11B+F4/80+ macrophages in the tumor microenvironment is reduced, thereby inhibiting tumor liver metastasis.
It significantly inhibits tumor liver metastasis, and improves the inhibitory effect on tumors by regulating the proportion of macrophages in the tumor microenvironment.
Smart Images
Figure HDA0005097684580000011 
Figure HDA0005097684580000012
Abstract
Description
Technical Field
[0001] The invention relates to the field of medical technology, and in particular to application of Lactobacillus brevis in inhibiting liver metastasis of tumor. Background Art
[0002] Many tumors are prone to metastasize to the liver, such as breast cancer, colorectal cancer, lung cancer, gastric cancer, etc. The main reasons why cancer is prone to metastasize to the liver are as follows: (1) The liver has a very rich blood flow and two blood supply systems. The liver is the main organ for nutrient metabolism and has a very rich blood supply. When tumors occur in organs such as the colorectum, lungs, and stomach, tumor cells can circulate in the blood and move to various organs. The liver has a very rich blood flow, so cancer cells can easily move to the liver with the blood, causing liver metastasis. (2) For tumors in the digestive system, blood flow first enters the liver. The venous return of abdominal organs such as the stomach, small intestine, colorectum, spleen, and pancreas will first pass through the superior mesenteric vein or the inferior mesenteric vein, then enter the portal vein, and finally enter the liver. Therefore, if cancer occurs in abdominal and pelvic organs, cancer cells will first enter the liver with venous blood. The liver is their first stop. (3) The liver is very rich in nutrients. The blood in the gastrointestinal tract will first return to the liver. All nutrients absorbed by the intestines, such as amino acids, fats, glucose, vitamins, electrolytes, etc., first enter the liver. Therefore, the liver is richest in nutrients. When cancer cells reach the liver, they can absorb nutrients from the liver, making it easier for cancer cells to grow. (4) Seed-soil theory. Some researchers believe that in addition to the rich blood supply to the liver, the liver's microenvironment is more suitable for the survival of tumor cells. At the same time, the immune system in the liver may not be comprehensive and is in an immune tolerance mode. Cancer cells in the liver cannot be completely eliminated by immune cells, making it easier for cancer cells to survive in the liver.
[0003] The liver is a common target organ for metastasis of many tumors, such as colorectal cancer, gastric cancer, and pancreatic cancer, and about 50% of solid tumors metastasize to the liver. Liver antigen-presenting cells, such as dendritic cells (DCs), Kupffer cells, and hepatic stellate cells, present antigens in an immune-tolerant mode, forming an immunosuppressive microenvironment suitable for the infiltration and colonization of circulating tumor cells, which is a common immune mechanism for liver metastasis in many tumors. However, since liver metastasis is often discovered in the late stage, surgical treatment is limited, and existing treatment methods such as targeted therapy and immunotherapy resistance have resulted in poor inhibition of tumor liver metastasis by conventional anti-tumor drugs. Summary of the invention
[0004] In view of the above problems, the present invention provides a drug for inhibiting tumor liver metastasis, which can significantly inhibit tumor liver metastasis.
[0005] The invention provides a medicine for inhibiting tumor liver metastasis, comprising pharmaceutically acceptable adjuvants and active pharmaceutical ingredients, wherein the active pharmaceutical ingredient is Lactobacillus brevis.
[0006] In one embodiment, the excipients include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.
[0007] In one embodiment, the dosage form of the drug includes capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.
[0008] The present invention also provides the use of Lactobacillus brevis in preparing a medicine for treating and / or preventing tumor liver metastasis.
[0009] In one embodiment, the drug for treating and / or preventing tumor liver metastasis further comprises pharmaceutically acceptable excipients and / or drugs compatible with Lactobacillus brevis.
[0010] In one embodiment, the dosage of Lactobacillus brevis is (4-6)×10 10 CFU / Kg.
[0011] In one embodiment, the administration route of the drug for treating and / or preventing tumor liver metastasis includes oral administration.
[0012] In one embodiment, the drug for treating and / or preventing tumor liver metastasis reduces CD11B in the tumor microenvironment + F4 / 80 + Macrophage ratio to inhibit tumor liver metastasis.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] The drug for inhibiting tumor liver metastasis of the present invention uses Lactobacillus brevis as the active ingredient of the drug, and the mouse experiment verifies that the drug can inhibit tumor liver metastasis by reducing CD11B in the tumor microenvironment. + F4 / 80 + The proportion of macrophages significantly inhibited tumor liver metastasis. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The results of the effect of Lactobacillus brevis on pancreatic cancer liver metastasis in Example 1 are shown, wherein A is a live imaging image of a small animal with pancreatic cancer liver metastasis, and B is a statistical graph of the fluorescence value of the live imaging of A, *P<0.05;
[0016] Figure 2 The effect of Lactobacillus brevis on pancreatic cancer liver metastasis CD11B in Example 1 +F4 / 80 + The results of the impact of the diagram, where C is the flow logic diagram related to tumor infiltrating macrophages; D and E are tumor infiltrating CD11B + F4 / 80 + Macrophage result chart and statistical chart, ***P<0.001. DETAILED DESCRIPTION
[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.
[0019] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.
[0020] Example 1
[0021] The tumor liver metastasis model was established by injecting mouse pancreatic cancer cells PAN02 into the portal vein. It can be understood that the tumor liver metastasis model can be established by using the mouse liver metastasis model establishment technology disclosed in the art.
[0022] 1. The mice with successful modeling were gavaged with PBS and Lactobacillus brevis (Lactobacillus brevis: purchased from Beina Biotechnology, strain number BNCC337373) for seven days before modeling and every day after modeling. The amount of PBS was 200ul / mouse and the amount of Lactobacillus brevis was 1*10 9 CFU / mouse (i.e. the dosage is 5*10 per kg body weight 10 CFU / Kg), the results showed that Lactobacillus brevis significantly inhibited liver metastasis, such as Figure 1 shown.
[0023] 2. Experimental operation.
[0024] Preheat a water bath to 42°C and set aside. Fill an ice box with ice, PBS, DMEM, and FBS, and place the digestive enzyme on ice to melt in advance.
[0025] Isolation and digestion of tumor tissue.
[0026] 1. Anesthesia: Anesthetize the mice with 4% chloral hydrate (1 ml syringe) at 10 μL / 1 g body weight (about 200 μL per mouse), and then bleed from the orbital vein.
[0027] 2. Disinfect the mouse, place it on a foam board to fix the tumor, and put it into a small dish (on ice).
[0028] 3. Wash the tumor with PBS and remove the fur, capsule, necrotic tissue and blood clots.
[0029] 4. Transfer to a new small dish and cut into pieces of about 1mm on ice. 3 During the digestion, add one or two drops of DMEM (to prevent the tissue from drying out too much), transfer with a Pasteur tube into a tube containing 30 ml of digestive enzyme + DMEM (labeled, marked with time), and place in a water bath to digest for about 1 hour.
[0030] 5. After digestion, transfer with a Pasteur tube and filter twice (first 70μm, add 1mL FBS to terminate digestion, then 40μm). When passing through the 70μm filter, use a 5ml syringe glue stick to grind the tissue (grind lightly, add while grinding, do not grind all of it through the filter, grinding too much will result in too much interstitial or cell fragments), and finally add DMEM to rinse and adjust the volume to 40mL.
[0031] 6. The obtained cell suspension was balanced with DMEM and centrifuged at 470g for 5 min. (Note the centrifuge rotor model and RCF mode).
[0032] 7. Wash cells: discard the supernatant with a Pasteur tube, then add 40ml 2% FBS + PBS + DNA enzyme (1μL for 1mL) and gently resuspend (add along the tube wall, remember not to pour directly), centrifuge at 470g, 5min. (Repeat this step 1-2 times depending on the situation).
[0033] 8. Remove impurities: discard the supernatant with a Pasteur tube, then add 40ml 2% FBS + PBS to gently resuspend (add along the tube wall, remember not to pour directly), centrifuge at 50g for 3min. Pipette the supernatant into another 50ml centrifuge tube, then centrifuge at 470g for 5min. (Repeat this step 1-2 times depending on the situation).
[0034] 9. Wash cells: discard the supernatant with a Pasteur tube, then add 40 ml of 2% FBS + PBS to gently resuspend (add along the tube wall, remember not to pour directly), centrifuge at 470g for 5 minutes.
[0035] 10. Lyse red blood cells: Prepare red blood cell lysis solution in advance (warm to room temperature). Add 3 ml according to the amount of cells, lyse for 5 minutes, neutralize with PBS, centrifuge at 470g for 5 minutes, and remove the supernatant. If there is still red at this time, lyse again (1-2 ml, 5 minutes).
[0036] If there are still a lot of cells after red blood cell lysis, repeat step 8 1-2 times.
[0037] 11. Staining. Collect and aliquot the cells, centrifuge at 500g for 5 minutes. Live or dead staining: wash once with D-PBS before staining, dye (FVS620) 1:1000 (D-PBS dilution), 15 minutes
[0038] Wash cells: add 2% FBS + PBS and gently resuspend, centrifuge at 470g for 5 minutes.
[0039] Prepare and stain antibodies: Blocking antibody + anti-cross-color antibody + required sorting antibody with PBS and stain
[0040] Wash cells: add 2% FBS + PBS and gently resuspend, centrifuge at 470g for 5 minutes.
[0041] PBS reselection flow cytometry test.
[0042] 3. Experimental results.
[0043] The experiment found that CD11B in the tumor microenvironment after Lactobacillus brevis treatment + F4 / 80 + The proportion of macrophages was significantly reduced, such as Figure 2 As shown in the literature published in the art, the ratio of macrophages / monocytes in liver metastasis tissue is significantly higher than that in non-metastatic tissue. Therefore, CD11B + F4 / 80 + The proportion of macrophages was significantly reduced, which means that Lactobacillus brevis regulates CD11B in the tumor microenvironment + F4 / 80 + Macrophage ratio to inhibit liver metastasis.
[0044] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0045] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A drug for inhibiting tumor liver metastasis, characterized in that: The invention comprises pharmaceutically acceptable auxiliary materials and active pharmaceutical ingredients, wherein the active pharmaceutical ingredient is Lactobacillus brevis.
2. The drug according to claim 1, characterized in that The auxiliary materials include at least one of solvents, disintegrants, flavoring agents, colorants, lubricants, antioxidants, preservatives, adhesives, fillers or thickeners.
3. The drug according to claim 1, characterized in that The dosage form of the drug includes capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.
4. Use of Lactobacillus brevis in the preparation of drugs for treating and / or preventing tumor liver metastasis.
5. The use according to claim 4, characterized in that: The drug for treating and / or preventing tumor liver metastasis also includes pharmaceutically acceptable adjuvants and / or drugs compatible with Lactobacillus brevis.
6. The use according to claim 4, characterized in that: The dosage of Lactobacillus brevis is (4-6)×10 10 CFU / Kg.
7. The use according to any one of claims 4 to 6, characterized in that: The administration route of the drug for treating and / or preventing tumor liver metastasis includes oral administration.
8. The use according to claim 7, characterized in that: The drug for treating and / or preventing tumor liver metastasis reduces CD11B in the tumor microenvironment + F4 / 80 + Macrophage ratio to inhibit tumor liver metastasis.