Application of astragalus polysaccharide in preparation of glioma treatment medicine

Abstract

The invention discloses application of astragalus polysaccharide in preparation of glioma treatment medicine, and particularly relates to the technical field of glioma treatment. The specific application is shown in two aspects: 1, as a natural medicine, the astragalus polysaccharide can promote polarization of macrophages to M1 type and inhibit polarization of macrophages to M2 type, so that the immunosuppression state in a glioma microenvironment is reversed, the growth of glioma is inhibited, and glioma cells can be killed; the astragalus polysaccharide has no obvious proliferation influence on macrophages and astrocytes, namely has no toxic or side effect on normal cells; secondly, the astragalus polysaccharide can directly inhibit dryness and proliferation of the glioma stem cells, promote apoptosis of the glioma stem cells and inhibit relapse of the glioma to a certain extent, and a new research thought and a medicine preparation method are provided for solving the problems that the glioma relapse is difficult to treat and the like. The astragalus polysaccharide shows a good application prospect for treating glioma.

Description

Technical Field

[0001] The present invention relates to the technical field of glioma treatment, and in particular to the application of astragalus polysaccharide in the preparation of glioma treatment drugs. Background Art

[0002] Glioma is the most common primary brain tumor in adults, accounting for 80% of brain tumors. Currently, the treatments for glioma mainly include radiotherapy, chemotherapy, surgical resection, and immunotherapy, which has become popular in recent years. However, due to the complexity of the central nervous system and the invasiveness of glioma, surgical resection is difficult, and the existence of glioma stem cells makes it difficult to completely remove the lesions. In addition, since the blood-brain barrier strictly controls the exchange of substances in the brain, chemotherapy drugs cannot reach effective concentrations in the tumor area, and the efficacy is poor. After treatment, patients are very likely to relapse, and their 5-year survival rate is less than 10%.

[0003] In the glioma immune microenvironment, a large number of immune cells are infiltrated. The interaction between immune cells and glioma cells forms a glioma immunosuppressive microenvironment. Among them, tumor-associated macrophages are the largest proportion of immune cells. There are two main polarization types, namely M1 tumor-associated macrophages and M2 tumor-associated macrophages. Among them, M1 tumor-associated macrophages inhibit tumor growth and metastasis, while M2 tumor-associated macrophages promote tumor growth and metastasis and promote the formation of an immunosuppressive microenvironment. In the glioma microenvironment, M1 accounts for only a minority, and the vast majority are M2 tumor-associated macrophages. Therefore, it is of great significance to find a natural drug that can reprogram the polarization direction of tumor-associated macrophages and inhibit the stemness-related functions of brain glioma stem cells.

[0004] Astragalus polysaccharide (APS) is a water-soluble heteropolysaccharide extracted, concentrated and purified from the dried roots of Astragalus mongolica or Astragalus membranaceus, a plant of the Leguminosae family. It is composed of hexuronic acid, glucose, fructose, rhamnose, arabinose, galacturonic acid and glucuronic acid. It can be used as an immunostimulant or regulator, and has antiviral, anti-tumor, anti-aging, anti-radiation, anti-stress and antioxidant effects[9].

[0005] At present, the treatment of glioma is still mainly radiotherapy, chemotherapy, and surgical resection. However, due to the existence of the blood-brain barrier, chemotherapy drugs or radiotherapy drugs cannot reach effective concentrations in the tumor area and thus cannot effectively treat glioma. The complexity of the central nervous system also makes surgical resection extremely difficult. Therefore, glioma has the characteristics of high lethality, high recurrence, and difficulty in curing.

[0006] In addition, there are the following technical difficulties: (1) Due to the existence of the immunosuppressive microenvironment of glioma, the human immune system cannot function normally and the treatment effect is not high. (2) Due to the existence of glioma stem cells, glioma patients are very likely to relapse after treatment. (3) Because existing chemotherapy drugs directly kill gliomas, they lead to the recruitment of myeloid-derived suppressor cells, further promoting the immune escape of gliomas and the formation of an immunosuppressive microenvironment, resulting in poor treatment efficacy for patients.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide the use of astragalus polysaccharide in preparing a glioma therapeutic drug.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a use of astragalus polysaccharide in the preparation of a drug for treating glioma.

[0011] After screening, the inventors found that astragalus polysaccharide, as a natural medicine, can promote macrophage polarization to M1 type and inhibit polarization to M2 type, thereby inhibiting the growth of glioma and killing glioma cells. Moreover, astragalus polysaccharide has no obvious proliferation effect on macrophages and astrocytes, that is, it has no toxic side effects on normal cells. Astragalus polysaccharide shows good application prospects in the treatment of glioma. In addition, the inventors also found that astragalus polysaccharide itself can directly inhibit the stemness and proliferation of glioma stem cells, promote the apoptosis of glioma stem cells, and can achieve a synergistic anti-tumor effect with macrophages polarized to the M1 subtype in vivo, providing new research ideas and drug preparation methods for solving the problems of glioma recurrence and difficult treatment.

[0012] In a preferred embodiment of the present invention, the above-mentioned drug has at least one of the following uses:

[0013] (1) Inducing macrophage polarization to M1 type, inhibiting the growth of glioma stem cells and killing them;

[0014] (2) inhibiting macrophage polarization toward M2 type;

[0015] and (3) inhibiting the stemness and proliferation of glioma stem cells and promoting the apoptosis of glioma stem cells.

[0016] "Stemness" refers to the self-renewal ability of stem cells. In a short period of time, Astragalus polysaccharide can achieve a highly effective killing and inhibitory effect on glioma stem cells.

[0017] In a preferred embodiment of the present invention, the above-mentioned drug has the following use: inhibiting the growth of glioma cells. Compared with low-concentration, short-term astragalus polysaccharide treatment, the effect of inhibiting differentiated glioma stem cells and glioma cells under high-concentration, long-term astragalus polysaccharide treatment is more obvious.

[0018] In a preferred embodiment of the present invention, the above-mentioned drug has the following use: inhibiting the volume of glioma. The above-mentioned inhibition of the volume of glioma means that compared with the control group not treated with astragalus polysaccharide, after the astragalus polysaccharide treatment, the volume of glioma is slightly decreased or significantly decreased, which is within the protection scope of the present invention.

[0019] In a preferred embodiment of the present invention, the above-mentioned drug has the following use: prolonging the life cycle of the test animal. The above-mentioned prolonging the life cycle of the test animal means that the life cycle of the glioma test animal treated with astragalus polysaccharide is prolonged to a certain extent or significantly prolonged compared with the glioma experimental animal not treated with astragalus polysaccharide, which is within the protection scope of the present invention.

[0020] The above-mentioned drugs also include combination drugs;

[0021] The combined drug is selected from temozolomide, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors, CYP17A1 inhibitors, saikosaponin F, saikosaponin G, saikosaponin C, saikosaponin A, saikosaponin D, camptothecin, podophyllotoxin, nitrosoureas, procarbazine and vinblastine chemotherapy drugs;

[0022] Preferably, the HMGCR inhibitor is selected from statins, and the statins are selected from simvastatin or fluvastatin; the CYP17A1 inhibitor is abiraterone acetate or a salt thereof.

[0023] In a preferred embodiment of the present invention, the above-mentioned drug further comprises a pharmaceutically acceptable carrier. The carrier includes, for example, a pharmaceutically acceptable additive or excipient, and the pharmaceutically acceptable additive or excipient is selected from one or more of the following: solvent, buffer, emulsifier, suspending agent, decomposing agent, disintegrant, dispersant, binder, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delay agent, flavoring agent, sweetener, pigment and liposome.

[0024] Excipients can be, for example, water, ethanol, 2-propanol, glycerol, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, glucose, fructose, lactose, sucrose, dextrose, molasses, starch, modified starch, gelatin, sorbitol, inositol, mannitol, microcrystalline cellulose, methylcellulose, carboxymethylcellulose, cellulose acetate, shellac, cetyl alcohol, polyvinyl pyrrolidone, paraffin, wax, natural and synthetic rubber, gum arabic, alginate, dextran, saturated and unsaturated fatty acids, stearic acid, magnesium stearate, zinc stearate, glyceryl stearate Oily esters, sodium lauryl sulfate, edible oils, sesame oil, coconut oil, peanut oil, soybean oil, lecithin, sodium lactate, polyoxyethylene and polyoxypropylene fatty acid esters, sorbitan fatty acid esters, sorbic acid, benzoic acid, citric acid, ascorbic acid, tannic acid, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, magnesium oxide, zinc oxide, silicon dioxide, titanium oxide, titanium dioxide, magnesium sulfate, zinc sulfate, calcium sulfate, potassium carbonate, calcium phosphate, dicalcium phosphate, potassium bromide, potassium iodide, talc, kaolin, pectin, crospovidone, agar and bentonite.

[0025] In a preferred embodiment of the present invention, the above-mentioned drugs also include drugs adjusted by structural optimization and modification. For example, the structural formula (C10H7ClN2O2S) of astragalus polysaccharide is optimized to make it easier to pass through the blood-brain barrier, and its structure can also be modified by polypeptides, etc. The selected polypeptides have blood-brain barrier permeability and glioma stem cell targeting.

[0026] In a preferred embodiment of the present invention, the dosage form of the above-mentioned drug is selected from tablets, capsules, granules, suspensions, powders or injections. Tablets are selected from sugar-coated tablets, film-coated tablets or enteric-coated tablets. Capsules are selected from hard capsules or soft capsules.

[0027] In a preferred embodiment of the present invention, the dosage form of the above-mentioned drug is selected from injection.

[0028] In a second aspect, the present invention also provides the use of astragalus polysaccharide in the preparation of a glioma stem cell inhibitor for inhibiting the stemness of glioma stem cells.

[0029] In a third aspect, the present invention also provides the use of astragalus polysaccharide in preparing a blood-brain barrier permeability drug.

[0030] In a preferred embodiment of the present invention, the blood-brain barrier permeability drug also includes a combination drug, which is selected from other drugs that easily pass through the blood-brain barrier, such as sulfonamides, nitroimidazoles, quinolones, and rifampicin. These drugs have high lipid solubility. In addition, there are penicillins, cefuroxime, third and fourth generation cephalosporins, cephalosporins, monocyclic β-lactams, carbapenems, glycopeptides, antibacterial drugs, etc.

[0031] The dosage form of the drug is selected from tablets, capsules, granules, suspensions, powders or injections.

[0032] The present invention has the following beneficial effects:

[0033] 1. Astragalus polysaccharide can promote macrophage polarization to M1 type and inhibit polarization to M2 type, thereby reversing the immunosuppressive microenvironment of glioma, inhibiting the growth of glioma and killing glioma cells.

[0034] 2. Astragalus polysaccharide has no obvious effect on the proliferation of macrophages and astrocytes, that is, it has no toxic side effects on normal cells.

[0035] 3. Astragalus itself can significantly inhibit the stemness and proliferation of glioma stem cells and promote the apoptosis of glioma stem cells.

[0036] 4. Astragalus polysaccharide itself has the effect of inhibiting the stemness and proliferation of glioma stem cells, which can achieve a synergistic anti-tumor effect with macrophages polarized to the M1 subtype in vivo, providing new research ideas and drug preparation methods for solving the problems of glioma recurrence and difficulty in treatment.

[0037] 5. Astragalus polysaccharide medicine does not contain other Chinese herbal extracts or Western medicines when it works. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 It is an overview diagram of the experimental methods involved in the experiments of the present invention;

[0040] Figure 2 It is the fluorescence quantitative PCR test results of different drugs in drug screening experiments;

[0041] Figure 3 This is a statistical diagram of the growth of macrophages and astrocytes after treatment with astragalus polysaccharide;

[0042] Figure 4 This is the experimental result of the effect of Astragalus polysaccharide on the polarization of mouse mononuclear macrophages;

[0043] Figure 5 This is the experimental result of the effect of Astragalus polysaccharide on the polarization of macrophages extracted from mouse bone marrow;

[0044] Figure 6This is a graph showing the effect of Astragalus polysaccharide on the growth of glioma cells or differentiated glioma stem cells;

[0045] Figure 7 This is the result diagram of the effect of Astragalus polysaccharide on the growth of glioma stem cells;

[0046] Figure 8 This is the result diagram of the effect of astragalus polysaccharide on the stemness of glioma stem cells;

[0047] Fig. 9 The figure shows the apoptosis results of glioma cells, astrocytes and glioma stem cells after treatment with astragalus polysaccharide;

[0048] Fig.10 This is the experimental result of Astragalus polysaccharide promoting macrophages to kill glioma cells;

[0049] Fig.11 This is a graph showing the experimental results that Astragalus polysaccharides can inhibit mouse tumor growth in vivo. DETAILED DESCRIPTION

[0050] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0051] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0052] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0053] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0054] Example 1

[0055] This example conducts a screening experiment of astragalus polysaccharide by summarizing the drug library of the School of Pharmacy of Henan University, firstly screening the literature, and then using real-time fluorescence quantitative PCR technology to screen out astragalus polysaccharide in the natural drug library. Figure 1 shown.

[0056] The specific experimental methods are as follows:

[0057] Mouse mononuclear macrophages were added with PBS, LPS (lipopolysaccharide) and IFN-γ (γ-interferon), IL-4 (interleukin-4) and IL-13 (interleukin-13), astragalus polysaccharide (APS), andrographolide, matrine, tuckahoe polysaccharide, baicalin, pexidartinib, and ficus polysaccharide to induce their polarization, among which LPS and IFN-γ are known M1 macrophage inducers, and IL-4 and IL-13 are known M2 macrophage inducers. After waiting for one day of drug treatment, the cells were harvested, the total cell RNA was extracted and reversely transcribed into cDNA, and the expression levels of M1 cell markers (CD86, TNF-α) under different treatments were detected by real-time fluorescence quantitative PCR to determine the direction of macrophage polarization induced by natural drugs.

[0058] Fluorescence quantitative PCR test results refer to Figure 2 As shown, Figure 2 As shown, A and B are the expression levels of M1 cell markers (CD86, TNF-α) under different treatments (compared with the expression levels of macrophages under PBS treatment, i.e., the Control group, with the Control group macrophages as the threshold).

[0059] Experimental results refer to Figure 2 The results showed that compared with other natural medicines, the content of M1 cell markers increased significantly after APS treatment, and the increase was comparable to that of known M1 macrophage inducers (LPS and IFN-γ), indicating that Astragalus polysaccharides can induce macrophage polarization to M1 cells.

[0060] Example 2

[0061] This example verifies the effect of Astragalus polysaccharide on the proliferation of astrocytes and macrophages.

[0062] HA1800 and ASCR are astrocytes, and Raw264.7 are mouse monocytes and macrophages.

[0063] The cell suspension after cell digestion was sampled and counted, and the density of cells diluted to an appropriate level was calculated. 150 μL of sterilized PBS buffer was added to each well around the edges of the 96-well plate to prevent the final results from being inaccurate due to evaporation of water in the experimental part of the central area. The 5 wells in the same column of the inner 15 wells were treated the same as parallel controls. In the 3 columns in the central area, only 1000 cells and only culture medium were added, respectively, and the two columns marked with added cells were drug addition (APS (800 μg / ml)) and blank control groups. The plates were placed in a 37°C incubator and cultured for 0 days, 1 day, 3 days, and 5 days, respectively. During the treatment, 10 μL of CCK-8 reagent was added to each well, wrapped in tin foil, and placed in a 37°C environment for 4 hours according to the staining depth. The OD value at 450 nm was measured using an enzyme marker.

[0064] Experimental results refer to Figure 3 The results showed that 800μg / ml Astragalus polysaccharide had no significant inhibitory effect on the growth of macrophages and astrocytes (P﹥0.05).

[0065] Example 3

[0066] This experimental example verifies the effect of Astragalus polysaccharide on the polarization of mouse mononuclear macrophages.

[0067] Mouse mononuclear macrophages were added with PBS, LPS (lipopolysaccharide) and IFN-γ (γ-interferon), 200, 400, 600, 800, 1000, 1500 μg / ml of astragalus polysaccharide (APS) to induce polarization, among which LPS and IFN-γ are known M1 macrophage inducers. After waiting for one day of drug treatment, the cells were harvested, total cell RNA was extracted and reversely transcribed into cDNA, and real-time fluorescence quantitative PCR was performed to detect the expression levels of M1 cell markers (CD86, MCP1, CD80, TNF-α) under different treatments to determine the optimal concentration of astragalus polysaccharide to induce macrophage polarization.

[0068] Experimental results refer to Figure 4 AD, the results showed that 800 μg / ml of astragalus polysaccharide can promote the polarization of macrophages to M1 type and inhibit their polarization to M2 type.

[0069] In addition, mouse mononuclear macrophages were added with PBS, LPS (lipopolysaccharide) and IFN-γ (γ-interferon), IL-4 (interleukin-4) and IL-13 (interleukin-13), and astragalus polysaccharide (APS) to induce their polarization, among which LPS and IFN-γ are known M1 macrophage inducers, and IL-4 and IL-13 are known M2 macrophage inducers. After waiting for one day of drug treatment, the cells were collected, and the total cell RNA was extracted and reversely transcribed into cDNA. Real-time fluorescence quantitative PCR was performed to detect the expression levels of M1 cell markers (CD86, TNF-α) under different treatments, and the expression levels of M2 cell markers (CD206, Arg-1) were used to determine the direction of macrophage polarization induced by astragalus polysaccharide.

[0070] Experimental results refer to Figure 4 As shown, Figure 4 Figures E and F are statistical graphs of the expression levels of M1 cell markers, G and H are statistical graphs of the expression levels of M2 cell markers, and E is a flow cytometry result graph. The horizontal axis is the staining result of FITC fluorescent CD206 antibody (M2 macrophage marker), and the vertical axis is the staining result of APC fluorescent CD86 antibody (M1 macrophage marker). The larger the upper left corner, the more the cell tends to M1 polarization, and the lower right corner indicates that the cell is polarized toward M2 cell type.

[0071] The experimental results showed that 800 μg / ml of Astragalus polysaccharide can promote the polarization of mouse monocytes and macrophages to M1 type and inhibit their polarization to M2 type.

[0072] Example 4

[0073] This experimental example verifies the effect of astragalus polysaccharide on the polarization of macrophages extracted from mouse bone marrow.

[0074] Macrophages extracted from mouse bone marrow were added to PBS, LPS (lipopolysaccharide) and IFN-γ (γ-interferon), IL-4 (interleukin-4) and IL-13 (interleukin-13), and astragalus polysaccharide (APS) to induce their polarization, among which LPS and IFN-γ are known M1 macrophage inducers, and IL-4 and IL-13 are known M2 macrophage inducers. After waiting for one day of drug treatment, the cells were collected, and the total cell RNA was extracted and reversely transcribed into cDNA. Real-time fluorescence quantitative PCR was performed to detect the expression levels of M1 cell markers (CD86, TNF-α) under different treatments, and the expression levels of M2 cell markers (CD206, IRF-4) were used to determine the direction of macrophage polarization induced by astragalus polysaccharide.

[0075] Experimental results refer to Figure 5As shown, 800 μg / ml of Astragalus polysaccharide can promote the polarization of mouse bone marrow-extracted macrophages to M1 type and inhibit their polarization to M2 type.

[0076] Example 5

[0077] This example verifies the effect of astragalus polysaccharide on the proliferation of glioma cells or differentiated glioma stem cells.

[0078] GL261 is a mouse glioma cell, U87 is a human glioma cell, and 83-Differentiation is a differentiated human glioma stem cell.

[0079] The cell suspension after cell digestion was sampled and counted, and the density of cells diluted to an appropriate level was calculated. 150 μL of sterilized PBS buffer was added to each well around the edges of the 96-well plate to prevent the final results from being inaccurate due to evaporation of water in the experimental part of the central area. The 5 wells in the same column of the inner 15 wells were treated the same as parallel controls. In the 3 columns in the central area, only 1000 cells and only culture medium were added, and the two columns marked with added cells were drug addition (APS (400, 800, 1000, 1500 μg / ml)) and blank control groups. The plates were placed in a 37°C incubator and cultured for 0 days, 1 day, 3 days, and 5 days (long-term), and 0 days, 1 day, and 3 days (short-term). During the treatment, 10 μL of CCK-8 reagent was added to each well, wrapped in tin foil, and placed in a 37°C environment for 4 hours according to the staining depth. The OD value at 450 nm was measured using an enzyme marker.

[0080] Experimental results refer to Figure 6 As shown, the results showed that 800 μg / ml Astragalus polysaccharide had no obvious inhibitory effect on the growth of glioma cells and differentiated glioma stem cells in a short period of time, but under long-term treatment, the growth of glioma cells and differentiated glioma stem cells was inhibited.

[0081] Example 6

[0082] This example explores the effect of astragalus polysaccharide on the proliferation of glioma stem cells.

[0083] X01, 83, 022, 528, 448 are human glioma stem cells, and 7080 is mouse glioma stem cell.

[0084] The cell suspension after cell digestion was sampled and counted, and the density of cells diluted to an appropriate level was calculated. 150 μL of sterilized PBS buffer was added to each well around the edges of the 96-well plate to prevent the final results from being inaccurate due to evaporation of water in the experimental part of the central area. The 5 wells in the same column of the inner 15 wells were treated the same as parallel controls. In the 3 columns in the central area, only 1000 cells and only culture medium were added, and the two columns marked with added cells were drug addition (APS (400, 800, 1000, 1500 μg / ml)) and blank control groups. The plates were placed in a 37°C incubator and cultured for 0 days, 1 day, 3 days, and 5 days (long-term), and 0 days, 1 day, and 3 days (short-term). During the treatment, 10 μL of CCK-8 reagent was added to each well, wrapped in tin foil, and placed in a 37°C environment for 4 hours according to the staining depth. The OD value at 450 nm was measured using an enzyme marker.

[0085] Experimental results refer to Figure 7 The results showed that astragalus polysaccharide could significantly inhibit the growth of glioma stem cells (human or mouse) regardless of whether they were treated for a long time or a short time, or at a high concentration or a low concentration.

[0086] Example 7

[0087] This example explores the effect of astragalus polysaccharide on the stemness of glioma stem cells.

[0088] After cell digestion, the cell suspension was sampled and counted, and the appropriate cell density was calculated. In a 96-well plate, every three columns were used as an area, and 100, 50, 25, and 5 cells were added to the four areas respectively, which were divided into two plates, namely the drug-added (APS (800μg / ml)) and the blank control group. The plates were placed in a 37°C incubator for 5 days (the edge of the 96-well plate was replenished with fluid on the third day). The number of spheres in each plate and each area was calculated.

[0089] Experimental results refer to Figure 8 The results showed that after treating glioma stem cells with 800 μg / ml of astragalus polysaccharide and PBS for 5 days, it was found that the stemness of glioma stem cells in the astragalus polysaccharide treatment group was significantly decreased compared with the control group, i.e., the PBS treatment group.

[0090] Example 8

[0091] This example investigates the apoptosis results of astrocytes, glioma cells and glioma stem cells after treatment with astragalus polysaccharide.

[0092] U251 is a glioma cell, HA1800 is an astrocyte, and X01 is a glioma stem cell.

[0093] The cells were divided into two parts and 800μg / ml of Astragalus polysaccharide and an equal amount of PBS were added to each part. After three days of treatment, the cells were collected for flow cytometry technology experiments. The horizontal axis is the staining result of FITC fluorescently labeled Annexin V, which can detect the externalization of phosphatidylserine, an important feature of cell apoptosis, and the vertical axis is the staining result of propidium iodide (PI) staining solution showing red fluorescence. Propidium iodide can stain necrotic cells or cells that have lost cell membrane integrity in the late stage of apoptosis.

[0094] Experimental results refer to Fig. 9 The results showed that after three days of treatment with 800 μg / ml Astragalus polysaccharide, glioma cells and astrocytes did not undergo apoptosis, while glioma stem cells underwent obvious apoptosis.

[0095] Example 9

[0096] Mouse mononuclear macrophages were treated with PBS, LPS (lipopolysaccharide), IFN-γ (γ-interferon), and astragalus polysaccharide (APS) for one day, and then the cells were collected. After the collected cells were co-cultured with glioma stem cells (X01-Luc) labeled with firefly luciferase (Luciferase) for 48 hours, the collected cells were tested for RLU (relative light unit) to determine the amount of glioma stem cells labeled with firefly luciferase (Luciferase).

[0097] Experimental results refer to Fig.10 , macrophages treated with 800 μg / ml of astragalus polysaccharide can kill glioma stem cells.

[0098] Example 10

[0099] Orthotopic tumor experiments were performed in mice.

[0100] Mice aged 6-8 weeks were selected and all mice were placed in a sterile environment with controlled temperature and humidity. 10w glioma stem cells (X01-Luc) were injected into the mouse brain. After the tumor model was successfully established, the mice were randomly divided into a blank group and an APS group (100mg / kg). The astragalus polysaccharide group was gavaged with 100mg / kg of astragalus polysaccharide solution, and the blank control group was gavaged with an equal amount of 0.9% saline. All mice were given the drug continuously for 20 days. During the experimental period, the mice were free to eat and drink. The weight and survival period of the mice were recorded. The experimental results refer to Fig.11 The results showed that after administration of astragalus polysaccharide, the weight loss rate of mice slowed down, the tumor volume decreased, and the survival period was prolonged.

[0101] In summary: Astragalus polysaccharide has no toxic side effects on macrophages and normal astrocytes, and has a certain inhibitory effect on differentiated glioma stem cells and glioma cells at high concentrations for a long time, but has a short-term efficient killing and inhibitory effect on glioma stem cells; at the same time, Astragalus polysaccharide can promote macrophage polarization to M1 type, thereby reversing the glioma immunosuppressive microenvironment, thereby killing glioma stem cells, achieving a 1+1>2 effect. In addition, the Astragalus polysaccharide used in the present invention does not contain other Chinese medicine extracts or Western medicines when exerting its medicinal effects.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0103] References:

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[0105] [2]Rhun EL, Preusser M, Roth P, et al. Molecular targeted therapy of glioblastoma[J]. Cancer Treatment Reviews, 2019,80:101896.

[0106] [3]Aldape K, Brindle KM, Chesler L, et al. Challenges to curing primary brain tumors[J]. Nature Reviews Clinical Oncology, 2019.

[0107] [4]Allen BM,Hiam KJ,Burnett CE,et al.Systemic dysfunction and plasticity of the immune macroenvironment in cancer models[J].Naturemedicine,2020(4):1-10.

[0108] [5] Quail DF, Joyce JA. The Microenvironmental Landscape of BrainTumors. Cancer Cell. 2017Mar 13;31(3):326-341.

[0109] [6]Orecchioni M, Ghosheh Y, Pramod AB, Ley K. Macrophage Polarization: Different Gene Signatures in M1(LPS+)vs.Classically and M2(LPS-)vs.Alternatively Activated Macrophages. Front Immunol. 2019May 24; 10:1084.

[0110] [7] Zhang Bo, Cao Yajuan, Wu Jianchun, et al. The role of tumor-associated macrophages in tumor invasion and metastasis and its regulation by traditional Chinese medicine [J]. Journal of Guangzhou University of Chinese Medicine, 2020, 37(4): 6.

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Claims

1. Application of Astragalus polysaccharide in the preparation of drugs for the treatment of glioma.

2. The use according to claim 1, characterized in that: The drug has at least one of the following uses: (1) Inducing macrophage polarization to M1 type, inhibiting the growth of glioma stem cells and killing them; (2) inhibiting macrophage polarization toward M2 type; and (3) inhibiting the stemness and proliferation of glioma stem cells and promoting the apoptosis of glioma stem cells.

3. The use according to claim 1, characterized in that: The drug has at least one of the following uses: (1) Inhibit the growth of glioma cells; (2) inhibiting the volume of glioma; (3) Prolong the life span of test animals.

4. The use according to claim 1, characterized in that The drugs also include combination drugs; The combined drug is selected from temozolomide, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors, CYP17A1 inhibitors, saikosaponin F, saikosaponin G, saikosaponin C, saikosaponin A, saikosaponin D, camptothecin, podophyllotoxin, nitrosoureas, procarbazine and vinblastine chemotherapy drugs; Preferably, the HMGCR inhibitor is selected from statins, and the statins are selected from simvastatin or fluvastatin; and the CYP17A1 inhibitor is abiraterone acetate or a salt thereof.

5. The use according to any one of claims 1 to 4, characterized in that: The medicament further includes a pharmaceutically acceptable carrier.

6. The use according to any one of claims 1 to 4, characterized in that: The drugs also include drugs that have been adjusted through structural optimization and modification.

7. Astragalus polysaccharide is used in the preparation of glioma stem cell inhibitors that inhibit the stemness of glioma stem cells.

8. Use of Astragalus polysaccharide in the preparation of blood-brain barrier permeability drugs.

9. The use according to claim 8, characterized in that The blood-brain barrier permeability drug also includes combined drugs.

10. The use according to any one of claims 1 to 4 or the use according to any one of claims 8 to 9, characterized in that: The dosage form of the drug is selected from tablets, capsules, granules, suspensions, powders or injections.

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