Use of psychotria asiatica in the preparation of a drug for treating glioma

Abstract

The application relates to the field of biological medicines, and particularly relates to application of bruceine in preparation of a medicine for treating neuroglioma. The bruceine is a small-molecule compound derived from brucea javanica. Researches show that the bruceine can inhibit proliferation, migration and invasion of glioma cells, and induce apoptosis of the glioma cells; meanwhile, the bruceine can inhibit growth of a subcutaneous tumor of a mouse glioma. Further researches show that the bruceine can interact with SHC-binding protein 1 (SHCBP1), and inhibit the malignant phenotype of the glioma cells by targeting the SHCBP1. In short, the application provides a new application of the small-molecule compound bruceine in resisting glioma, and provides a new candidate medicine for treating neuroglioma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of croton extract in the preparation of drugs for treating glioma. Background Technology

[0002] Gliomas are among the most common primary tumors of the central nervous system, accounting for approximately 40% to 50% of all primary central nervous system tumors, and representing a significant proportion of malignant central nervous system tumors. According to the World Health Organization's classification of central nervous system tumors, gliomas can be divided into different malignancy grades. Glioblastoma (GBM) is highly malignant, characterized by rapid growth, strong invasiveness, high recurrence rate, and poor prognosis, resulting in a low 5-year survival rate and posing a serious threat to patients' lives and health.

[0003] Currently, the clinical treatment of gliomas mainly adopts a comprehensive treatment model combining surgical resection, radiotherapy, and temozolomide chemotherapy. However, for high-grade gliomas, due to the diffuse infiltrative growth of tumor cells and the difficulty in accurately identifying tumor boundaries, surgery often cannot completely remove the tumor cells. Furthermore, some glioma cells are resistant to radiotherapy and chemotherapy, and the dose-limiting toxicity of temozolomide can lead to bone marrow suppression (such as severe thrombocytopenia and neutropenia), often accompanied by gastrointestinal reactions such as nausea and vomiting. In addition, dexamethasone, routinely used to relieve postoperative cerebral edema, may increase the risk of infection and affect survival prognosis. Therefore, even with aggressive treatment, the overall efficacy of existing glioma treatment regimens still needs improvement, and there is an urgent need to find new glioma treatment options or drugs to provide more choices for clinical practice.

[0004] Bruceantin, also known as Yadan Ting or Yadan Ding, has the chemical formula C64. 28 H 36 O 11 The compound, with CAS number 41451-75-6, is a small molecule compound derived from the traditional Chinese medicine Brucea javanica. Existing studies have shown that Brucea javanica has anti-leukemia and antiprotozoal activities, but its anti-tumor pharmacological effects and targets in glioma have not yet been reported. Summary of the Invention

[0005] The technical problem to be solved by this invention is that the overall efficacy of existing treatments for glioma is not good, and a new application of the small molecule compound crotonin in the treatment of glioma is provided, giving more options for clinical practice.

[0006] The purpose of this invention is to provide the use of croton extract in the preparation of a medicament for treating glioma.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: This invention combines in vitro experiments with multiple glioma cell lines. Results showed that crotonin significantly inhibited the proliferation, migration, and invasion of glioma cells and induced apoptosis. In vivo experiments established a glioma model by subcutaneous injection of glioma cells into the axilla of C57BL / 6J mice. Results showed that crotonin inhibited the growth of subcutaneous gliomas in mice, significantly reducing glioma volume and weight. This indicates that crotonin has a good anti-glioma effect. Further research showed that crotonin targets and inhibits the malignant phenotype of glioma cells through its interaction with SHCBP1, exhibiting a significant anti-glioma therapeutic effect.

[0008] The crotonin used in this invention is a small molecule compound of the quassin class, prepared from croton seeds. Crotonin possesses a wide range of pharmacological activities, and its structural formula is shown in formula (Ⅰ) below: .

[0009] Therefore, the following applications of the croton extract of this invention are all within the scope of protection of this invention: The application of croton extract in the preparation of drugs for treating glioma.

[0010] Furthermore, the glioma is a glioblastoma.

[0011] Furthermore, the glioma is an astrocytoma.

[0012] Specifically, the drug treats glioma by reducing the proliferative capacity of glioma cells.

[0013] The drug treats gliomas by reducing the migration and invasion capabilities of glioma cells.

[0014] Application of croton extract in the preparation of drugs that induce apoptosis in glioma cells.

[0015] Application of croton extract in the preparation of drugs that reduce the activity of glioma cells.

[0016] Furthermore, the drug treats glioma by targeting SHCBP1. SHC-binding and spindle-associated 1 (SHCBP1) is a protein involved in cell mitosis and spindle formation, and is closely related to cell cycle regulation and cell proliferation. Previous studies have suggested that SHCBP1 is abnormally expressed in various tumors and may participate in the proliferation, migration, and invasion of tumor cells. High expression of SHCBP1 in gliomas is closely related to poor prognosis, suggesting that SHCBP1 may be a potential intervention target for glioma treatment. The results of this invention show that croton extract can exert its anti-glioma effect by targeting and binding to SHCBP1, providing a new drug option for glioma treatment.

[0017] Application of croton extract in the preparation of drugs that reduce the volume and weight of gliomas in tumor-bearing mice.

[0018] Furthermore, the dosage form of the drug is an injection.

[0019] The present invention has the following beneficial effects: This invention provides a novel application of crotonin in the preparation of drugs for treating glioma. In vitro experiments show that crotonin significantly reduces the proliferation, migration, and invasion of glioma cells, while also inducing apoptosis. In vivo experiments show that crotonin inhibits the growth of subcutaneous glioma tumors in mice. This indicates that crotonin has a good anti-glioma effect. This invention provides a new candidate drug for the treatment of glioma. Attached Figure Description

[0020] Figure 1 The IC50 curve shows the inhibitory effect of croton extract on glioma cells (U87MG, U251, U118, SF126, LN18, T98G, LN229, GL261). 50 .

[0021] Figure 2 The results of the glioma cell cloning experiment are shown in the top image (top) and the statistical chart of the glioma cell cloning experiment is shown in the bottom image (bottom).

[0022] Figure 3 The results of the Transwell migration experiment are shown in the top figure and the statistical chart of the Transwell migration experiment results are shown in the bottom figure.

[0023] Figure 4 The image shows the results of the Transwell invasion experiment (top) and the statistical graph of the Transwell invasion experiment results (bottom).

[0024] Figure 5The graph shows the results of apoptosis detection by flow cytometry (top) and the statistical graph shows the results of apoptosis detection by flow cytometry (bottom).

[0025] Figure 6 This study compares the therapeutic effects of croton extract on a subcutaneous tumor-bearing model of C57BL / 6J mice (GL261). Figure 6 A is a growth curve of mouse tumor volume; B is a graph of the final size of mouse tumor; C is a statistical graph of mouse tumor weight.

[0026] Figure 7 This is a flowchart of the DARTS combined mass spectrometry analysis mode.

[0027] Figure 8 Volcano plot of differentially expressed proteins in DARTS samples.

[0028] Figure 9 The image shows the results of Western blot detection of SHCBP1 expression in DARTS samples.

[0029] Figure 10 The results of Western blot analysis of SHCBP1 expression in the samples (top) and the grayscale statistics of SHCBP1 expression (bottom) are shown.

[0030] Figure 11 The figure shows the knockdown efficiency of SHCBP1 detected by Western blot.

[0031] Figure 12 This is a statistical chart of cell viability testing.

[0032] Figure 13 This is a statistical graph of apoptosis experiments.

[0033] Quantitative results are expressed as mean ± standard deviation (SD). All data were statistically analyzed using GraphPad Prism (GraphPad Software, San Diego, USA). Data on tumor volume changes over time were analyzed using two-way ANOVA, with appropriate multiple comparison corrections applied according to the experimental design. Tumor weight was analyzed using an unpaired Student's t-test. All bar charts were generated using GraphPad Prism.* P <0.05,** P <0.01, *** P <0.001, **** P <0.0001, P <0.05 is statistically significant. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] The male C57BL / 6J mice used in this embodiment were purchased from Zhuhai Beston Biotechnology Co., Ltd.; U87MG, U251, U118, SF126, LN18, T98G, LN229 cells were purchased from Wuhan Pronosei Life Science Co., Ltd.; GL261 cells were purchased from Wuhan Shangen Biotechnology Co., Ltd.; and yabatin was purchased from Chengdu Kloma Biotechnology Co., Ltd.

[0037] Example 1: Detection of the effect of croton extract on glioma cell viability using the CCK-8 assay 1. Method (1) Glioma cells in logarithmic growth phase (U87MG, U251, U118, SF126, LN18, T98G, LN229, GL261, etc.) were digested and counted. 5 × 10³ cells were seeded per well in a 96-well plate with a culture volume of 100 μL. Three replicates were set up for each group, along with blank wells containing only culture medium and no cells.

[0038] (2) After the cells adhered to the culture vessel overnight, the original culture medium was removed and complete culture medium containing different concentrations (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μM) of croton was added and the cells were treated for 48 hours.

[0039] (3) After the treatment time is up, remove the culture medium from the well and add 100 μL of fresh complete culture medium containing 10 μL of CCK-8 reagent to each well. Incubate in a 37℃, 5% CO2 incubator for 1 hour in the dark.

[0040] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of each well at a wavelength of 450 nm.

[0041] (5) The relative survival rate of glioma cells was calculated using the following formula: Relative cell survival rate (%) = (A experimental group - A blank group) / (A solvent control group - A blank group) × 100%. Data were processed using Graphpad software. A dose-response curve was plotted with the logarithm of the crotonine concentration on the x-axis and the relative cell survival rate on the y-axis. The half-maximal inhibitory concentration (IC50) of crotonine on glioma cells was calculated. 50 .

[0042] 2. Results Experimental results are as follows Figure 1 As shown, croton extract can significantly inhibit the activity of glioma cells, IC50. 50 The concentration ranged from 0.016 μM to 0.137 μM, indicating significant anti-glioma cell activity.

[0043] Example 2: Colony formation assay to detect the effect of crotonin on the long-term proliferative capacity of glioma cells. 1. Method (1) Take LN229 cells in the logarithmic growth phase, digest and count them, and seed 1000 cells per well into a 6-well plate, adding 2 mL of complete culture medium. Gently shake the culture plate to disperse the cells evenly. Incubate the cells overnight at 37°C in a 5% CO2 incubator to allow the cells to adhere fully to the plate.

[0044] (2) After cell adhesion, discard the original culture medium and treat the cells with complete culture medium containing different concentrations (0, 0.01 μM, 0.1 μM) of crotonin for 10–14 days, with 3 replicates for each concentration group. Replace the culture medium with fresh complete culture medium containing the drug every 5 days during the culture period. Observe the cell colony formation daily. When visible cell colonies appear in the control group, and most colonies contain more than 50 cells, the culture is terminated.

[0045] (3) After the culture is completed, discard the culture medium, wash the cells twice with PBS, add 1 mL of 4% paraformaldehyde fixative to each well, and fix at room temperature for 20 minutes.

[0046] (4) After discarding the fixative, wash twice with PBS. Add 1 mL of 0.1% to 0.5% crystal violet staining solution to each well and stain at room temperature in the dark for 20 minutes.

[0047] (5) After staining, discard the staining solution and rinse with water until the background of the well plate is clear. Invert the culture plate and let it air dry at room temperature.

[0048] (6) Take a photograph of the entire culture plate and observe the cell clones using an inverted microscope. Define an independent cell colony consisting of no less than 50 cells as a valid clone. Use ImageJ software to count the number of valid clones formed in each well. The relative colony formation rate is calculated according to the following formula: Relative colony formation rate (%) = Number of clones in the croton-treated group / Number of clones in the solvent control group × 100%.

[0049] 2. Results Experimental results are as follows Figure 2 As shown, compared with the control group, the number of cell clones formed in the croton-treated group was reduced, and the clone area was smaller, showing a certain concentration-dependent relationship. The results indicate that croton treatment can inhibit the colony-forming ability and long-term proliferation ability of glioma cells.

[0050] Example 3: Transwell assay to detect the effect of crotonin on the migration and invasion ability of glioma cells. 1. Method (1) Transwell migration experiment: 1) A 24-well Transwell chamber with an 8 μm pore size was used. 500 μL of DMEM high-glucose medium containing 10% fetal bovine serum was added to the lower chamber as chemotactic conditions for cell migration.

[0051] 2) Take U87MG cells in the logarithmic growth phase and pretreat them for 12 h with the corresponding concentrations (0, 0.1 μM, 1 μM) of crotonin prepared in serum-free medium. Set up 3 replicates for each group.

[0052] 3) After treatment, cells were washed and digested with PBS and collected. The cells were resuspended in serum-free DMEM high-glucose medium, and 100 μL of cell suspension was added to the upper chamber of a Transwell container, containing 1 × 10⁶ cells per chamber. 5 Cells. Appropriate concentrations (0.1 μM, 1 μM) of crotonin were added to the upper and lower chamber culture media. The Transwell plates were incubated at 37°C in a 5% CO2 incubator for 6 h.

[0053] 4) After the culture is complete, remove the Transwell chamber and discard the culture medium in the upper chamber. Fix the chamber in 4% paraformaldehyde for 20 minutes.

[0054] 5) After washing with PBS to remove 4% paraformaldehyde, stain with 0.1%~0.25% crystal violet solution for 20 minutes.

[0055] 6) Rinse with water or PBS to remove crystal violet solution, and gently wipe away any unmigrated cells from the membrane surface with a moistened cotton swab. Air dry at room temperature.

[0056] 7) Use the EVOS M7000 imaging system to photograph the migrating cells on the submembrane surface and use ImageJ software to count the cells. Five fields of view were randomly selected from the EVOS M7000 imaging results for photographing and counting. The average number of migrating cells in each field of view was used to represent cell migration ability, and the relative migration rate was calculated: Relative migration rate (%) = Number of migrating cells in the experimental group / Number of migrating cells in the solvent control group × 100%.

[0057] (2) Transwell invasion experiment: 1) Melt the Matrigel at 4°C overnight, maintaining a low temperature throughout the experiment. Prepare the Matrigel working solution using pre-cooled serum-free medium at a ratio of 100:1.

[0058] 2) Add 100 μL of diluted Matrigel evenly to the surface of the Transwell upper chamber membrane with an 8 μm pore size, and incubate in a 37°C incubator for 1 hour to allow the matrix gel to solidify.

[0059] 3) Collect U87MG cells in the logarithmic growth phase and pretreat them for 12 h with appropriate concentrations (0, 0.1 μM, 1 μM) of crotonin prepared in serum-free medium. After washing with PBS, digest and collect the cells. Resuspend the cells in serum-free DMEM high-glucose medium, count and adjust the cell concentration, and set up 3 replicates for each group.

[0060] 4) Add 100 μL of cell suspension to the upper chamber of the Matrigel-coated cell container, and seed each chamber with 1×10⁶ cells. 5 Cells were added. 500 μL of complete culture medium containing 10% fetal bovine serum was added to the lower chamber to provide chemotactic conditions for cell invasion. The same concentration of crotonin was added to both the upper and lower chambers according to the groupings, ensuring a consistent final solvent concentration across all groups.

[0061] 5) Place the Transwell culture plate in a 37℃, 5% CO2 incubator and incubate for 6 h.

[0062] 6) After culturing, remove the Transwell chamber, fix the cells in the Transwell chamber and stain them with crystal violet. The specific steps are the same as those for the migration experiment.

[0063] 7) Use the EVOS M7000 imaging system to photograph the invasive cells on the submembrane surface and use ImageJ software to count the cells. Five fields of view were randomly selected from the EVOS M7000 imaging results for photographing and counting. The average number of invasive cells in each field of view was used to represent the cell invasion ability, and the relative invasion rate was calculated: Relative invasion rate (%) = Number of invasive cells in the experimental group / Number of invasive cells in the solvent control group × 100%.

[0064] 2. Results Experimental results are as follows Figures 3-4 As shown. Compared with the control group, the number of U87MG cells crossing the Transwell membrane was reduced in the croton-treated group ( Figure 3 The number of cells passing through Matrigel was also significantly reduced. Figure 4 The above results indicate that crotonin can inhibit the migration and invasion of glioma cells.

[0065] Example 4: Annexin V-FITC / PI double staining method for detecting croton-induced apoptosis in glioma cells 1. Method (1) Glioma cells U87MG in the logarithmic growth phase were digested, counted, and then distributed at 4 × 10⁴ cells per well.5 One cell was seeded in a 6-well plate and cultured overnight in an adherent state.

[0066] (2) The next day, add complete culture medium containing different concentrations (0, 0.1 μM, 1 μM) of croton, and place the cells in a 37°C, 5% CO2 incubator for 24 hours. Each group has at least 3 replicates.

[0067] (3) After the drug treatment is completed, collect the floating cells in the culture supernatant of each well and digest and collect the adherent cells.

[0068] (4) Centrifuge at 300×g for 5 minutes and discard the supernatant. Wash the cells with pre-cooled PBS to collect them.

[0069] (5) The Annexin V-FITC / PI staining kit was purchased from KGI Biotechnology, product batch number KGA108. The cells were resuspended in 100 μL of 1× binding buffer, and 1 μL of Annexin V-FITC and 1 μL of propidium iodide staining solution were added to each tube and mixed gently.

[0070] (6) Incubate at room temperature in the dark for 5 minutes. After incubation, add 400 μL of 1× binding buffer to each tube, mix well, and then use flow cytometry to detect the cells within 1 hour.

[0071] 2. Results Experimental results are as follows Figure 5 As shown, compared with the solvent control group, treatment with crotonin can induce apoptosis in glioma cells in a dose-dependent manner.

[0072] Example 5: Evaluation of the inhibitory effect of crotonin on glioma growth using a mouse subcutaneous xenograft tumor model. 1. Method (1) Fourteen male SPF-grade C57 mice, aged 5 weeks and weighing about 20g, were purchased and acclimatized for one week in the SPF-grade animal room of the Experimental Animal Center of Sun Yat-sen University Shenzhen Campus.

[0073] (2) Expand the culture of GL261 glioma cells, digest and count them on the day of inoculation, and slowly inject 100 μL of cell suspension into the subcutaneous tissue of the right axilla of mice using a 1 mL sterile syringe. Each mouse is inoculated with 5 × 10⁶ cells. 6 We used glioma cells to construct a subcutaneous glioma xenograft model.

[0074] (3) Observe the condition of mice and the inoculation site daily after cell inoculation. When a palpable subcutaneous mass appears at the inoculation site, measure the long and short diameters of the tumor using calipers. When the average tumor volume reaches approximately 80-120 mm... 3At that time, the subcutaneous xenograft tumor model was considered successfully established. Based on tumor volume, the tumor-bearing mice were divided into a control group and a croton extract treatment group, with 7 mice in each group. (4) In the treatment group, the croton extract was diluted with normal saline to the appropriate concentration, while in the control group, only normal saline was injected. The drug was administered via intraperitoneal injection at a dose of 3 mg / kg, once every 3 days for 21 consecutive days.

[0075] (5) During the experiment, observe the condition of the mice. Measure the long and short diameters of the tumor using calipers every 3 days and record the animal's weight. The tumor volume is calculated using the following formula: Tumor volume (mm²) 3 = Major axis × Minor axis² ÷ 2. Where the major axis is the maximum diameter of the tumor, and the minor axis is the maximum diameter perpendicular to the major axis.

[0076] (6) Plot tumor growth curves for each group of mice with administration time on the x-axis and tumor volume on the y-axis. Mice were euthanized the day after the last administration, and the subcutaneous tumor tissue was completely dissected, photographed, and weighed. Experimental data are expressed as mean ± standard deviation. Data on tumor volume changes over time were analyzed using a two-way ANOVA, with appropriate multiple comparison corrections applied according to the experimental design. Tumor weight was analyzed using an unpaired Student's t-test. * P <0.05,** P <0.01, * P A value <0.05 is considered statistically significant.

[0077] 2. Results Experimental results are as follows Figure 6 As shown, the subcutaneous xenograft volume in the model control group mice continued to increase over time, while the tumor growth rate in the croton-treated group mice decreased compared to the control group. Figure 6 (A). A schematic diagram of the tumor at the experimental endpoint and a statistical graph of tumor weight show that treatment with crotonin significantly inhibited the growth of subcutaneous tumors. Figure 6 (B~C). The above results indicate that crotonin can inhibit the growth of subcutaneous xenografts formed by glioma cells in mice.

[0078] Example 6: DARTS combined with mass spectrometry analysis to screen potential targets for action of croton oil. 1. Method A schematic diagram of the experimental procedure is shown below. Figure 7 As shown, it includes the following steps: (1) Use M-PER™ mammalian protein extraction reagent, protease inhibitor, and phosphatase inhibitor to prepare cell lysis working solution and store on ice.

[0079] (2) Prepare a 10×TNC buffer containing 500 mmol / L Tris-HCl, 500 mmol / L NaCl and 100 mmol / L CaCl2, with a pH of 8.0; dilute with sterile ultrapure water to a 1×TNC buffer before use, which contains 50 mmol / L Tris-HCl, 50 mmol / L NaCl and 10 mmol / L CaCl2.

[0080] (3) Take U87MG glioma cells in the logarithmic growth phase, discard the culture medium, wash with PBS, and then add an appropriate amount of the above fine lysis working solution. Collect the cell lysis buffer using a cell scraper and transfer the lysis buffer to a pre-cooled centrifuge tube.

[0081] (4) Place the cell lysis buffer on ice for 20-30 minutes, gently mixing it every 5 minutes. Then centrifuge at 4°C and about 12000×g for 10-15 minutes, collect the supernatant, and obtain the total protein lysis buffer of glioma cells.

[0082] (5) Add an appropriate amount of 10×TNC buffer to the protein lysis buffer to make the final concentration of TNC buffer 1×.

[0083] (6) The protein concentration was detected by BCA method, and the protein concentration of each sample was adjusted to be consistent using lysis buffer, preferably to 2~5 mg / mL.

[0084] (7) Divide the protein into two groups, add croton extract and control DMSO respectively, and incubate at room temperature with gentle shaking for 1 hour.

[0085] (8) Divide the incubated proteins into groups and add them to the protein at Pronase ratios of 2000:1, 1000:1, 800:1, and 400:1, respectively. Add Pronase to the proteins and incubate at room temperature for 15 minutes. Prepare Pronase fresh using 1×TNC buffer. (9) After incubation, immediately add 5×SDS-PAGE loading buffer to make the final concentration 1×, and heat at 98℃ for 10 minutes for denaturation treatment.

[0086] (10) The denatured samples were digested with enzymes and detected by high performance liquid chromatography-tandem mass spectrometry system to identify the differences in protein abundance between the croton-treated group and the control group.

[0087] (11) The expression of SHCBP1 was detected by Western Blot experiment to confirm the binding effect of yabatin on SHCBP1.

[0088] 2. Results Figure 8The differential protein volcano plot shows the differentially expressed proteins that were upregulated and downregulated in the Bruceantin-treated group compared to the control group in the DARTS experiment. Figure 9 Western blotting analysis of the protective effect of Bruceantin on the protein SHCBP1 in DARTS samples showed that, compared with the control group, the Bruceantin treatment group slowed down the degradation of SHCBP1. This preliminarily indicates that Bruceantin binds to SHCBP1.

[0089] Example 7: CETSA experiments demonstrate that SHCBP1 is the target of croton extract. 1. Method (1) Take U87MG glioma cells in the logarithmic growth phase and treat them with high concentration of crotonin and an equal volume of DMSO for 1 hour.

[0090] (2) After treatment, discard the culture medium, wash the cells with PBS, then digest and collect the cells.

[0091] (3) After centrifuging the cells at about 300×g for 5 minutes, discard the supernatant and wash twice with pre-cooled PBS.

[0092] (4) The two groups of cells (control group and croton-treated group) were resuspended in pre-cooled PBS containing protease inhibitors and divided into 10 groups. Each group was heated at different temperatures: 40℃, 43℃, 46℃, 49℃, 52℃, 55℃, 58℃, 61℃, 64℃ and 67℃, respectively, for 3 minutes in a PCR instrument, and then immediately cooled at room temperature for 3 minutes.

[0093] (5) After heating and cooling, the cells in each tube were lysed. The cells were lysed by repeated freeze-thaw cycles in liquid nitrogen, with the freeze-thaw cycles repeated 3 times to ensure that the cells were fully ruptured.

[0094] (6) After lysis, the sample was centrifuged at 4°C and 12,000 rpm for 20 minutes to remove cell debris.

[0095] (7) Carefully aspirate the supernatant, add an appropriate amount of SDS-PAGE loading buffer, and heat at 95°C for 10 minutes to fully denature the protein.

[0096] (8) Western blot was used to detect the expression of SHCBP1 in the samples. ImageJ software was used to measure the gray value of SHCBP1 protein bands at each temperature point. The gray value of SHCBP1 at 40℃ was set to 100%, and the relative expression level of soluble SHCBP1 at each temperature point was calculated. Graphpad software was used to plot the thermal stability curves of SHCBP1 in the solvent control group and the croton treatment group with temperature as the abscissa and the relative remaining amount of SHCBP1 as the ordinate.

[0097] 2. Results Experimental results are as follows Figure 10 As shown, croton can slow down the degradation of the protein SHCBP1 by high temperature, indicating that croton has a binding effect on SHCBP1 and can protect and slow down the degradation of the stable SHCBP1 protein.

[0098] Example 8: Detecting whether knocking down the target SHCBP1 alleviates the inhibitory effect of croton extract on glioma cells. 1. Method (1) Human glioma cells U87MG in the logarithmic growth phase were seeded in cell culture plates. When the cell confluence reached about 50% to 70%, they were infected with negative control interfering RNA lentivirus (shNC) and interfering RNA lentivirus targeting SHCBP1 (sh-SHCBP1), respectively.

[0099] (2) After 48 hours of infection and culture, the protein expression level of SHCBP1 was detected by Western blot to confirm the knockdown efficiency of SHCBP1.

[0100] (3) After SHCBP1 knockdown, cells from each group (shNC, shSHCBP1) were seeded in 96-well plates. After cell attachment, croton extract or an equal volume of solvent was added for 48 hours. The absorbance of each group of cells at 450 nm was measured according to the aforementioned CCK-8 assay, and the relative cell viability was calculated. Each group was set up with no less than 5 replicates, and the experiment was independently repeated at least 3 times.

[0101] (4) After SHCBP1 knockdown, cells were seeded in 6-well plates and treated with crotonin or solvent for 24 hours according to the above grouping. Suspended cells and adherent cells in the culture supernatant were collected and stained according to the Annexin V-FITC / PI double staining method described above. The early apoptosis rate, late apoptosis rate and total apoptosis rate of each group were detected by flow cytometry.

[0102] 2. Results Experimental results are as follows Figure 11 The results showed that SHCBP1 was significantly knocked down in the shSHCBP1 group; Figures 12-13The results showed that, compared with control cells, the inhibitory effect of crotonin on cell viability and its pro-apoptotic effect were significantly weakened in SHCBP1 knockdown cells. This indicates that the inhibitory effect of crotonin on glioma cell viability and its pro-apoptotic effect are partially dependent on the target SHCBP1.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of croton extract in the preparation of drugs for treating glioma, characterized in that, The structural formula of the Yadanting is shown in the following formula (Ⅰ): 。 2. The application according to claim 1, characterized in that, The glioma is an astrocytoma.

3. The application according to claim 1, characterized in that, The glioma in question is a glioblastoma.

4. The application according to claim 1, characterized in that, The drug treats gliomas by reducing the proliferative capacity of glioma cells.

5. The application according to claim 1, characterized in that, The drug treats gliomas by reducing the migration and invasion capabilities of glioma cells.

6. The application according to claim 1, characterized in that, The drug treats gliomas by inducing apoptosis in glioma cells.

7. The application according to claim 1, characterized in that, The drug treats gliomas by reducing the viability of glioma cells.

8. The application according to claim 7, characterized in that, The drug treats gliomas by targeting SHCBP1, which binds to glioma cells.

9. The application according to claim 1, characterized in that, The drug treats gliomas by reducing their size and weight.

10. The application according to claim 1, characterized in that, The drug is in the form of an injection.

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