Application of hyodeoxycholic acid and malignant glioma resisting medicine

Abstract

The invention relates to application of hyodeoxycholic acid and a malignant glioma resisting medicine, and relates to the technical field of biological medicine. The invention relates to an application of hyodeoxycholic acid, which is used for preparing a medicine for resisting malignant glioma. Human brain astroblastoma U87-MG cells are intervened by applying hyodeoxycholic acid, and through a CCK-8 cell viability experiment, a Transwell cell migration experiment and a cell apoptosis experiment, it is found that hyodeoxycholic acid can effectively inhibit growth and migration of the human brain astroblastoma U87-MG cells and promote U87-MG cell apoptosis, so that malignant progression of neuroglioma is inhibited, and the effect of treating neuroglioma is achieved. A new medicine is provided for treatment of glioma, and the application prospect is good.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of hyodeoxycholic acid and a drug for resisting malignant glioma. Background Art

[0002] Glioma is the most common primary tumor of the central nervous system, accounting for 81% of central nervous system malignant tumors, and mainly originates from glial tissue. According to the 2021 World Health Organization (WHO) classification of central nervous system tumors, gliomas are divided into 4 grades. Among them, glioblastoma (GBM), as a grade 4 glioma, is widely recognized as the most common type of primary brain malignant tumor, with the characteristics of high invasiveness, poor prognosis, and easy recurrence. At present, clinical treatments for gliomas include surgical resection, chemotherapy, immunotherapy, etc. Despite this, these treatments for malignant brain gliomas still seem inadequate, and the 5-year overall survival rate of patients with grade 4 gliomas is very low. Therefore, new and effective treatment strategies for malignant brain gliomas are urgently needed.

[0003] Hyodeoxycholic acid is an organic compound that inhibits bile acid formation and fat dissolution, lowering blood cholesterol and triglycerides. It is suitable for treating type Ia or Ib hyperlipidemia and atherosclerosis. It also has a certain antibacterial effect against Bordetella pertussis, Corynebacterium diphtheriae, and Staphylococcus aureus. It can also be used as an anti-inflammatory drug to treat chronic bronchitis and pediatric viral upper respiratory tract inflammation. Despite this, the role of hyodeoxycholic acid in glioblastoma remains unclear, and its anti-glioma activity has not been reported in the literature. There is no record of its use as an active ingredient in anti-glioma research or clinical applications. In view of this, the present invention provides a use of hyodeoxycholic acid and a drug for treating malignant gliomas. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an application of hyodeoxycholic acid and a drug for treating malignant gliomas. The purpose is to inhibit the growth and migration of glioma cells and promote the apoptosis of glioma cells by hyodeoxycholic acid, thereby providing a new drug and method for the prevention and treatment of gliomas.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In the first aspect, a use of hyodeoxycholic acid is used to prepare a drug for treating malignant glioma.

[0007] Furthermore, the hyodeoxycholic acid inhibits glioma by inhibiting the growth and / or migration of human brain glioblastoma cells.

[0008] Furthermore, the hyodeoxycholic acid promotes apoptosis of human brain astroglioblastoma cells to inhibit glioma.

[0009] Among them, hyodeoxycholic acid ( Hyo d eoxyc h o li c Acid) is a secondary bile acid with a high content in pig bile. It is the main active ingredient in pig bile in traditional Chinese medicine. It is referred to as HDCA and its molecular formula is C 24 H 40 O4, molecular weight is 392.57, CAS number is 83-49-8, and the structural formula is as follows:

[0010]

[0011] Furthermore, the half inhibitory concentration IC of hyodeoxycholic acid in human brain astroglioma U87-MG cells in vitro is 50 It is 617.3μM.

[0012] In a second aspect, a drug for treating malignant glioma comprises the hyodeoxycholic acid in the aforementioned application of hyodeoxycholic acid.

[0013] Furthermore, the drug also includes a pharmaceutically acceptable carrier and / or auxiliary components.

[0014] Furthermore, the drug further comprises at least one of a filler, a lubricant, a dispersant, a wetting agent, a binder, a regulator, a solubilizer, an antioxidant, an antibacterial agent, an emulsifier, and a disintegrant.

[0015] Furthermore, the dosage form of the drug is a clinically acceptable pharmaceutical preparation. The drug of the present invention can be prepared as a pharmaceutical composition according to methods well known in the art, and can be prepared into any dosage form suitable for human or animal use by combining the drug of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0016] Furthermore, the dosage form of the drug includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, and emulsions.

[0017] The actual dosage of the active ingredient (hyodeoxycholic acid of the present invention) in the pharmaceutical composition of the present invention should be determined based on a variety of relevant factors, including the severity of the disease to be treated, the route of administration, the age, gender, and weight of the patient. Therefore, the above dosage should not limit the scope of protection of the present invention in any way.

[0018] The beneficial effects of the present invention are as follows: the present invention uses hyodeoxycholic acid to intervene in human brain astroglioma U87-MG cells, and through CCK-8 cell viability experiments, Transwell cell migration experiments and cell apoptosis experiments, it is found that hyodeoxycholic acid can effectively inhibit the growth and migration of human brain astroglioma U87-MG cells, promote U87-MG cell apoptosis, and further inhibit the malignant progression of glioma, providing a new drug for the treatment of glioma and having good application prospects.

[0019] The present invention discloses the use of hyodeoxycholic acid in the preparation of anti-malignant glioma drugs for the first time. This study experimentally verified that hyodeoxycholic acid can significantly inhibit glioma cell growth and promote glioma cell apoptosis. This is unrelated to its known uses (anti-inflammatory, antibacterial, etc.), and no other existing compounds provide relevant inspiration. However, it possesses outstanding substantive characteristics, represents a significant advancement in the preparation of anti-malignant glioma drugs, and expands its new clinical indications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing that hyodeoxycholic acid inhibits the activity of U87-MG cells in an embodiment of the present invention;

[0021] Figure 2 Figure 1 is a graph showing that hyodeoxycholic acid inhibits U87-MG cell migration in an embodiment of the present invention; A is a cell image corresponding to the cell migration experiment; B is the statistical result of the number of migrating cells, and the statistical results are expressed as the mean ± standard deviation of three independent experiments, ***P<0.001, ****P<0.0001;

[0022] Figure 3 Figure 2 is a graph showing that hyodeoxycholic acid promotes apoptosis of U87-MG cells in an embodiment of the present invention; wherein A is a flow cytometry result corresponding to the cell apoptosis experiment; B is the statistical result of the cell apoptosis rate, and the statistical results are expressed as the mean ± standard deviation of three independent experiments, NS is not statistically significant, ****P<0.0001. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0024] Example

[0025] 1. Test materials and instruments.

[0026] 1.1 Test materials.

[0027] Human astroglioma U87-MG cells (purchased from Wuhan Punosai Biotechnology Co., Ltd.); MEM complete medium (purchased from Gibco); fetal bovine serum FBS (purchased from Sigma); 0.25% trypsin digestion solution (purchased from Gibco); penicillin-streptomycin mixture (purchased from Wuhan Punosai Biotechnology Co., Ltd.); hyodeoxycholic acid (HDCA, purchased from MedChemExpress (MCE) Company, catalog number: HY-N0169); DMSO (purchased from Sigma); CCK-8 detection kit (purchased from White Shark Biotechnology Co., Ltd.); 1% crystal violet dye (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.); Transwell chamber (8 μM) PC membrane (purchased from corning); Annexin V-FITC / PI cell apoptosis double staining kit (purchased from BD).

[0028] 1.2 Test equipment.

[0029] Experimental instruments: CO2 cell culture incubator (model Thermo3111; manufacturer Thermo Fisher Scientific); fully automatic cell counter (model CellDrop BF; manufacturer Denovix); microplate reader (model Spark; manufacturer TECAN); inverted fluorescence microscope (model CK X53; manufacturer OLYMPUS); flow cytometer (model NovoCyte; manufacturer Agilent).

[0030] 2. Test method.

[0031] 2.1 Cell culture and treatment.

[0032] Dissolve 100 mg of HDCA (hyodeoxycholic acid) in 5.09 mL of DMSO to obtain a 50 mM HDCA solution. Dilute HDCA with MEM complete medium (containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin) to final concentrations of 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, and 800 μM, respectively.

[0033] U87-MG cells were cultured in a 37°C, 5% CO2 cell culture incubator. When the cell density reached 80-90%, the cells were digested with 0.25% trypsin. After the cells were observed to be rounded under a microscope, complete culture medium was added to terminate the digestion and the cells were counted.

[0034] 2.2 Detection methods for cell viability assay, Transwell cell migration and flow cytometry apoptosis assay.

[0035] (1) Cell viability assay:

[0036] In this example, the effect of HDCA on the viability of U87-MG cells was detected by CCK-8 assay.

[0037] U87-MG cells in the logarithmic growth phase were seeded into 96-well plates with 100 μl of cell suspension per well and a cell number of 8 × 10 3 Cells were pre-cultured in a 37°C, 5% CO2 incubator for approximately 24 hours. Different concentrations of HDCA were added to each well and incubated for an additional 48 hours before cell viability testing.

[0038] Assess cell viability using the CCK-8 assay kit: Add 10 μl of CCK-8 reagent to each well and gently shake the plate to mix, taking care to avoid creating bubbles. Return the plate to the incubator and incubate for another 1 hour. Measure the absorbance (OD) at 450 nm using a microplate reader. Calculate the following formula: Cell viability = [(As-Ab) / (Ac-Ab)] × 100%;

[0039] Wherein, As: absorbance of experimental well (containing cells, culture medium, CCK-8 solution and drug solution);

[0040] Ac: absorbance of control well (containing cells, culture medium, and CCK-8 solution, but no drug);

[0041] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0042] The results are as follows Figure 1 shown.

[0043] (2) Cell migration assay:

[0044] In this example, the effect of HDCA on the migration ability of U87-MG cells was detected by Transwell cell migration assay.

[0045] Serum-free MEM medium was used to adjust the density of U87-MG cells in the logarithmic growth phase to 2 × 10 5Cells were plated at 100 μl of the cell suspension (100 μl / mL) into a Transwell chamber (upper chamber) and 800 μl of complete culture medium in the lower chamber. Different concentrations of HDCA (0 μM, 400 μM, 600 μM, and 800 μM) were added. Avoid introducing bubbles during manipulation. After incubation at 37°C in a 5% CO2 incubator for 24 hours, different concentrations of HDCA were added to each well. The cells were cultured for an additional 48 hours before testing.

[0046] Remove the Transwell chamber and wash twice with 1x PBS. Fix the cells with 4% paraformaldehyde for 10 minutes at room temperature. Discard the fixative, wash twice with PBS, and stain with 1% crystal violet for 15 minutes. After washing two to three times with PBS, gently remove any unmigrated cells from the upper layer with a cotton swab. Cells that have migrated to the lower layer of the microporous membrane were photographed using an inverted fluorescence microscope. Five randomly selected fields of view were photographed for each sample, and the number of migrated cells was counted using ImageJ.

[0047] (3) Cell apoptosis experiment:

[0048] In this example, flow cytometry apoptosis assay was used to detect the effect of HDCA on the apoptosis ability of U87-MG cells.

[0049] U87-MG cells in the logarithmic growth phase were seeded into 6-well plates with 2 mL of cell suspension per well and a cell number of 2 × 10 5 Each well was plated with different HDCA concentrations (0 μM, 400 μM, 600 μM, and 800 μM). The cells were cultured in a 37°C, 5% CO2 incubator for approximately 24 hours. Different concentrations of HDCA were added to each well and incubated for another 48 hours before apoptosis assay.

[0050] Digest the cells with EDTA-free trypsin and collect the cells by centrifugation at 2000 rpm for 5 minutes at room temperature. Resuspend the cells in pre-chilled 1× PBS and wash by centrifugation at 2000 rpm for 5 minutes. Add 100 μl of 1× Binding Buffer to resuspend the cells. Add 5 μl of Annexin V-FITC, mix well, and incubate at room temperature for 15 minutes in the dark. Add 5 μl of PI staining solution 5 minutes before loading the flow cytometer. Before loading the flow cytometer, add 400 μl of 1× Binding Buffer. Detect cell apoptosis using flow cytometry.

[0051] 2.3 Statistical analysis

[0052] The experiments were repeated three times, and the results are expressed as the mean ± SD of three independent experiments. Statistical analysis was performed using GraphPad Prism software 8.0 (GraphPad, California, USA). Differences between groups were assessed using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test. P < 0.05 was considered statistically significant, with *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.

[0053] 3. Experimental results.

[0054] The results are as follows Figure 1-3 As shown:

[0055] (1) By Figure 1 It was found that HDCA inhibited the viability of U87-MG cells. The effects of HDCA treatment at 0μM, 100μM, 200μM, 300μM, 400μM, 500μM, 600μM, 700μM, and 800μM on U87-MG cell viability were examined. CCK-8 assay results showed that cell viability decreased in a concentration-dependent manner with increasing HDCA treatment concentrations, reaching 50% at a treatment concentration of 617.3μM.

[0056] (2) From Figure 2 As shown in Figures A and B, HDCA effectively inhibits U87-MG cell migration. The effects of 400μM, 600μM, and 800μM HDCA on U87-MG cell migration were examined. The results of the cell migration assay showed that the number of cells migrating to the lower chamber decreased with increasing HDCA concentration.

[0057] (3) From Figure 3 As shown in Figures A and B, HDCA effectively promotes apoptosis in U87-MG cells. The effects of 400μM, 600μM, and 800μM HDCA on the apoptosis of U87-MG cells were examined. The results of the apoptosis assay showed that the apoptosis rate of tumor cells gradually increased with increasing HDCA concentration.

[0058] In summary, the present invention found through CCK-8 cell viability assay, Transwell cell migration assay and flow cytometry assay that HDCA can effectively inhibit the growth and migration of human brain glioblastoma U87-MG cells, promote the apoptosis of U87-MG cells, and thus inhibit the malignant progression of glioma, providing a new drug for the treatment of glioma and having good application prospects.

[0059] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An application of hyodeoxycholic acid, characterized in that, Used for preparing drugs for treating malignant glioma.

2. The use of hyodeoxycholic acid according to claim 1, characterized in that: The hyodeoxycholic acid inhibits glioma by inhibiting the growth and / or migration of human brain glioblastoma cells.

3. The use of hyodeoxycholic acid according to claim 1, characterized in that: The hyodeoxycholic acid promotes the apoptosis of human brain astroglioblastoma cells to inhibit glioma.

4. The use of hyodeoxycholic acid according to claim 1, characterized in that: The half inhibitory concentration IC of hyodeoxycholic acid in human astroglioma U87-MG cells in vitro 50 It is 617.3μM.

5. A drug for treating malignant glioma, characterized in that: The medicine comprises the hyodeoxycholic acid in the use of hyodeoxycholic acid according to any one of claims 1 to 4.

6. The drug for treating malignant glioma according to claim 5, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or auxiliary components.

7. The drug for treating malignant glioma according to claim 5, characterized in that: The medicine further comprises at least one of a filler, a lubricant, a dispersant, a wetting agent, a binder, a regulator, a solubilizer, an antioxidant, an antibacterial agent, an emulsifier, and a disintegrant.

8. The drug for treating malignant glioma according to claim 5, characterized in that: The dosage form of the drug is a clinically acceptable pharmaceutical preparation.

9. The drug for treating malignant glioma according to claim 8, characterized in that: The dosage form of the drug includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, and emulsions.

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