New application of formononetin in macrophages
By increasing the expression of CD86 protein on the surface of macrophages, reducing the expression of CD206 protein, polarizing M2 macrophages into M1 type, and using cyperidin in anti-tumor drugs, the macrophage pole in the prior art was solved The deficiency of chemistry and tumor suppression has achieved effective inhibition and safe drug effects on tumors.
Patent Information
- Application Number
- CN202411436002.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-15
- Publication Date
- 2025-01-17
AI Technical Summary
The lack of application of cynomolin for macrophage polarization in the prior art, especially in the tumor microenvironment, has failed to effectively utilize its potential against tumors.
By increasing the expression of CD86 protein on the surface of macrophages, reducing the expression of CD206 protein, M2 macrophages are polarized into M1 macrophages, and using cyperonidin as an active ingredient in antitumor drugs, including its The effective concentration of acceptable salts, esters, hydrates or combinations and auxiliary materials is 5 μg/mL to 10 μg/mL.
The inhibitory effect on tumors was achieved. The ability of cynomolin was verified in vitro and in vivo experiments, which significantly increased the M1/M2 ratio of macrophages, inhibited the growth of solid tumors in mice, and demonstrated high dose safety. sex.
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Figure CN119320748A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to a new use of formononetin in macrophages. Background Art
[0002] Macrophages are a type of immune cell that is widely found in various tissues and organs of the body. They originate from monocytes, which in turn originate from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytic cells that participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates.
[0003] Macrophages include M1 macrophages and M2 macrophages. M1 macrophages, also known as pro-inflammatory macrophages, function by producing nitric oxide, reactive oxygen species and a variety of inflammatory factors (such as TNF-α, IL-1β, etc.), while M2 macrophages, also known as anti-inflammatory macrophages, function by producing anti-inflammatory factors, growth factors, angiogenic factors and other molecules. They occupy a dominant position in the tumor microenvironment, promote the survival, proliferation and metastasis of tumor cells by secreting a variety of promoting molecules, and can inhibit the functions of other immune cells, such as inhibiting the activity of T cells and NK cells, thereby participating in immunosuppressive reactions.
[0004] Macrophages are key cells in the immune system, and their polarization and functional state have an important impact on the body's immune response and disease progression.
[0005] Formononetin is derived from the inflorescence and flowering branches and leaves of the leguminous plant Trifolium Pratense L. There is no report on the polarization effect of formononetin on macrophages. Summary of the invention
[0006] The technical problem to be solved by the present invention is to provide a new use of formononetin and related products thereof.
[0007] In order to solve the above technical problems, the technical solution provided by the present invention is: application of formononetin in macrophage polarization.
[0008] Preferably, the formononetin increases the expression of CD86 protein on the surface of macrophages and decreases the expression of CD206 protein, wherein formononetin polarizes M2 macrophages into M1 macrophages.
[0009] The second aspect of the present invention discloses a use of formononetin in anti-tumor drugs.
[0010] Preferably, formononetin is used as an active ingredient in the medicine.
[0011] Preferably, the drug comprises formononetin and pharmaceutically acceptable salts, esters, hydrates or combinations thereof of formononetin and excipients.
[0012] Preferably, the effective concentration of formononetin in the drug is 5 μg / mL to 10 μg / mL.
[0013] The advantages of the present invention over the prior art are as follows: the present invention provides a new use of formononetin, by which the expression of CD86 protein on the surface of macrophages is increased and the expression of CD206 protein is reduced, thereby polarizing M2 macrophages toward M1 macrophages, thereby achieving tumor inhibition. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 The experimental results show Figure 1 .
[0015] Figure 2 The experimental results show Figure 2 .
[0016] Figure 3 It is a schematic diagram of in vivo drug efficacy test. DETAILED DESCRIPTION
[0017] The following is combined with Figure 1-2 The present invention is described in further detail.
[0018] Cell viability assay
[0019] 1) 4T1 cells in the logarithmic growth phase were seeded into a 96-well plate, 3000 cells were added to each well, and the plate was cultured overnight in a cell culture incubator (37° C.) with 5% CO 2 .
[0020] 2) Adding drugs: Set up blank control wells (only culture medium), solvent control wells (complete culture medium + the same volume of solvent), and drug treatment groups (different concentrations of drugs for corresponding time, pay attention to the preparation of drug solution concentration).
[0021] 3) After the drug treatment time is up, add 10 μL of CCK-8 reagent to each well and incubate in an incubator for 2-4 hours. Place the 96-well plate on an oscillator for 5 minutes and measure the absorbance at 450 nm using an enzyme-labeled instrument.
[0022] Experimental results: At a low concentration of 10-20ug / ml, formononetin had no obvious inhibitory effect on the activity of mouse bone marrow-derived macrophages.
[0023] Macrophage polarization assay:
[0024] (1) Extraction: Healthy BALB / c male mice aged 10-12 weeks were killed and immersed in 75% medical alcohol for 3 min. The femurs and tibias of both lower limbs were then removed in a clean bench, and the fascia, muscle, and fat connected to the bones were removed.
[0025] (2) Collection: Cut open the femoral head or tibial head with ophthalmic scissors, use a 1 mL syringe to draw up PBS to blow out the cells in the bone marrow, collect the PBS suspension of mononuclear cells and centrifuge at 4°C, 300 g for 5 min.
[0026] (3) Red blood cell lysis: Take the precipitate and add 3 mL of red blood cell lysis buffer to lyse the red blood cells. After centrifugation, wash once with PBS.
[0027] (4) Induction: Place the extracted cells in DMEM medium containing 10% FBS and add 20 ng / mL M-CSF to promote cell differentiation into BMDM. Plate the cells in a 6-well plate and add 3×10 5 Place the 6-well plate containing cells in a 37°C incubator containing 5% CO2.
[0028] (5) Inducing differentiation: After four days, the cells gradually adhere to the wall, and the culture medium is replaced on the fourth day.
[0029] (6) Resting: On the seventh day, bone marrow-derived stem cells were differentiated into M0 macrophages and placed in DMEM complete medium for one day. Then, IL-4 20 ng / mL and IL-13 20 ng / mL were added for induction for 48 h.
[0030] (7) 5 μg / mL, 10 μg / mL formononetin or an equal volume of PBS were added to the in vitro induced M2-like BMDM and the cells were treated for 48 h.
[0031] (8) Prepare single cell suspension: discard the culture medium, wash twice with PBS, scrape the cells from the culture dish with a cell scraper, and resuspend in 50 μL running buffer.
[0032] (9) Staining: Add 50 μL running buffer and add flow cytometry antibodies CD86-AP, D206-PE, BV421-F4 / 80, CD11b-APC / Cy7, and CD16 / 32 at a volume ratio of 1:200 between flow cytometry antibody staining solution and running buffer. Place in a 4°C refrigerator and stain for 30 min away from light.
[0033] On-line: Centrifuge, remove the supernatant, add 400μL running buffer, place the sample on ice away from light, and wait for on-line testing
[0034] Results: Low concentration of formonectin was selected for in vitro administration, and it was found that it could effectively increase the expression of CD86 protein on the surface of macrophages, reduce the expression of CD206 protein, and had no significant change on the total number of macrophages, but had a significant improvement on the M1 / M2 subpopulation ratio. Therefore, formonectin can effectively polarize primary macrophages derived from the bone marrow of M2-like mice, and it was found in basic medical research that it can effectively test the growth inhibitory effect on a variety of mouse solid tumors.
[0035] In vivo efficacy test
[0036] The efficacy of formononetin on the mouse triple-negative breast cancer 4T1 subcutaneous tumor model.
[0037] Total number of mice: (18) balb / c (administered ip every other day)
[0038] Grouping: M group, low dose, high dose
[0039] Drug: Formononetin, the solvent is first the mother liquid DMSO, then corn oil; the volume is 200ul.
[0040] Treatment interval: When the tumor tissue grows to 4*5mm, medication can be started, which is about the 7th day. Tumor volume and mouse weight are measured every two days. Dosage dosage is as follows:
[0041] The low concentration of F was 20ng / mouse / 100ul, and the high concentration of F was 10mg / kg, i.e. 200ug / mouse / 100ul. The Model group was intraperitoneally injected with normal saline.
[0042] During the in vivo efficacy test, the tumor volume of mice was measured, and it was found that high-dose formononetin could effectively inhibit the growth of 4T1 triple-negative breast cancer in mice, while low-dose drugs could not effectively suppress tumors.
[0043] The mice were weighed at the end of the experiment, and it was found that the tumor weight of the mice in the high-dose group was significantly lower than that in the model group and the low-dose group.
[0044] However, regardless of high or low doses, the drug did not significantly inhibit the weight of mice, indicating that the drug was safe for mice at high and low doses ( Figure 3 ).
[0045] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.
[0046] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.
Claims
1. Application of formononetin in macrophage polarization.
2. The use according to claim 1, characterized in that: The formononetin increases the expression of CD86 protein on the surface of macrophages and reduces the expression of CD206 protein.
3. The use according to claim 2, characterized in that: The formononetin polarizes M2 macrophages into M1 macrophages.
4. An application of formononetin in anti-tumor drugs.
5. The use according to claim 4, characterized in that: In the medicine, formononetin is used as an active ingredient.
6. The use according to claim 4, characterized in that: The medicine comprises formononetin and pharmaceutically acceptable salts, esters, hydrates or combinations thereof of formononetin and auxiliary materials.