Use of cucurbitacin Q1 in the preparation of a medicament for treating glioma

Abstract

This invention relates to the application of cucurbitacin Q1 in the preparation of drugs for treating gliomas, belonging to the field of biomedical technology. The cucurbitacin Q1 of this invention has a significant inhibitory effect on glioma cell activity. It can inhibit the protein expression of JAK1, JAK2, and STAT4 by regulating the JAK-STAT signaling pathway, and block the phosphorylation activation of STAT3 protein, thus weakening downstream signal transduction in this pathway. Simultaneously, it can also exert an anti-tumor effect by regulating the phosphorylation level of related proteins in the PI3K-AKT signaling pathway. The cucurbitacin Q1 of this invention not only significantly inhibits the proliferation, invasion, and migration of glioma cells, but also significantly increases the apoptosis rate of glioma cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of cucurbitacin Q1 in the preparation of drugs for the treatment of glioma. Background Technology

[0002] Gliomas are among the most common primary malignant tumors of the central nervous system, characterized by high invasiveness, high recurrence rate, and extremely poor prognosis. The 2021 WHO Classification of Tumors of the Central Nervous System classifies gliomas into grades 1-4, with grade 4 glioblastoma (GBM) being the most prevalent and malignant subtype. Even with standard comprehensive treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, clinical benefit remains limited, and overall patient survival is poor. Currently, safe and effective treatment options are lacking, indicating a significant unmet medical need. Summary of the Invention

[0003] The purpose of this invention is to address the current lack of effective treatments for gliomas by providing a novel pharmaceutical approach for treating gliomas. To this end, this invention provides the application of cucurbitacin Q1 in the preparation of drugs for treating gliomas.

[0004] This invention provides the use of cucurbitacin Q1 in the preparation of a drug for treating glioma, wherein the chemical structural formula of cucurbitacin Q1 is shown in Formula I. , Formula I.

[0005] Preferably, the glioma is a brain glioma.

[0006] Preferably, the glioma is a glioblastoma.

[0007] Preferably, the treatment for glioma includes at least one of the following: ① to ④ ① Inhibit the proliferation of glioma cells; ②Inhibit the migration of glioma cells; ③ Inhibit the invasion of glioma cells; ④ Induces apoptosis in glioma cells.

[0008] Preferably, the drug comprises a liquid formulation, a solid formulation, or a spray formulation.

[0009] Preferably, cucurbitacin Q1 is the sole active ingredient of the drug.

[0010] Preferably, the working concentration of cucurbitacin Q1 in the drug is 0.001 μM to 25 μM.

[0011] This invention also provides the application of cucurbitacin Q1 in the preparation of products that regulate the JAK-STAT signaling pathway and / or regulate the PI3K-AKT signaling pathway.

[0012] Preferably, the regulation of the JAK-STAT signaling pathway includes at least one of the following: 1) Inhibit the expression of at least one of JAK1, JAK2, and STAT4 proteins; 2) Inhibit the phosphorylation activation of STAT3 protein.

[0013] Preferably, the regulation of the PI3K-AKT signaling pathway includes regulating the phosphorylation level of related proteins in the PI3K-AKT signaling pathway.

[0014] Beneficial effects: This invention provides the application of cucurbitacin Q1 in the preparation of drugs for treating gliomas. The chemical structural formula of cucurbitacin Q1 is shown in Formula I. Cucurbitacin Q1 of this invention exhibits significant inhibitory effects on glioma cell activity. It can inhibit the protein expression of JAK1, JAK2, and STAT4 by regulating the JAK-STAT signaling pathway and blocking the phosphorylation activation of STAT3 protein, thereby weakening downstream signal transduction in this pathway. Simultaneously, it can also exert anti-tumor effects by regulating the phosphorylation levels of related proteins in the PI3K-AKT signaling pathway. Cucurbitacin Q1 of this invention not only significantly inhibits the proliferation, invasion, and migration of glioma cells but also significantly increases the apoptosis rate of glioma cells. As verified by examples, the cucurbitacin Q1 described in this invention exhibits significant anti-glioma activity even at extremely low concentrations (0.001 μM~25 μM), showing a concentration-dependent inhibitory effect on the proliferation of human glioma U251 cells and effectively reducing tumor cell viability. Furthermore, the cucurbitacin Q1 described in this invention can significantly inhibit the migration and invasion of glioma cells, and the inhibitory effect increases with increasing drug concentration. Simultaneously, it can effectively induce glioma cell apoptosis, significantly increasing the tumor cell apoptosis rate. In summary, the cucurbitacin Q1 used in this invention possesses potent anti-tumor activity at low concentrations and exhibits low cytotoxicity to normal nerve cell SVGP12, demonstrating good selectivity. It provides a novel candidate drug for the clinical treatment of gliomas and possesses good development value and clinical application prospects. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 Figure 1 shows the cell viability detection results of U251 cells after proliferation culture under different concentrations of cucurbitacin Q1 intervention (n=3); among them, This indicates that P < 0.01. This indicates that P < 0.0001; Figure 2 Figure 1 shows the cell viability detection results of SVG-P12 cells after proliferation culture under different concentrations of cucurbitacin Q1 intervention (n=3); among them, This indicates that P < 0.05; Figure 3 Representative graphs showing the migration rate of U251 cells under different concentrations of cucurbitacin Q1 intervention (n=3). Figure 4 The figure shows the migration rate of U251 cells under different concentrations of cucurbitacin Q1 intervention (n=3); among them, This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001; Figure 5 The graph represents the results of U251 cell migration count detection under different concentrations of cucurbitacin Q1 intervention. Figure 6 A statistical graph showing the migration number of U251 cells under different concentrations of cucurbitacin Q1 intervention (n=3); among them, This indicates that P < 0.0001; Figure 7 Representative graphs showing the detection results of U251 cell invasion numbers under different concentrations of cucurbitacin Q1 intervention (n=3); Figure 8 A statistical graph showing the results of U251 cell invasion counts under different concentrations of cucurbitacin Q1 intervention (n=3); among them, This indicates that P < 0.0001; Figure 9 A representative graph showing the apoptosis rate of U251 cells under cucurbitacin Q1 intervention (n=3). Figure 10 A statistical graph showing the apoptosis rate of U251 cells after cucurbitacin Q1 intervention (n=3); among them, This indicates that P < 0.05. This indicates that P < 0.01; Figure 11 Figure I (n=3) shows the results of Western blotting detection of cucurbitacin Q1 protein expression levels after administration. Figure 12 Figure I shows the statistical results of Western blotting (WB) detection of cucurbitacin Q1 protein expression levels after administration (n=3). Figure 12 In the figure, A represents the statistical results of JAK1 protein expression level detection after cucurbitacin Q1 administration; Figure 12 In the figure, B is a statistical graph showing the results of JAK2 protein expression level detection after cucurbitacin Q1 administration; Figure 12 In the figure, C represents the statistical results of p-STAT3 protein expression level detection after cucurbitacin Q1 administration; Figure 12 In the figure, D represents the statistical results of STAT3 protein expression level detection after cucurbitacin Q1 administration; Figure 12 E in the figure represents the statistical results of STAT4 protein expression level detection after cucurbitacin Q1 administration; This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001; Figure 13 Figure II (n=3) shows the results of Western blotting detection of cucurbitacin Q1 protein expression levels after administration. Figure 14 Figure II shows the statistical results of Western blotting (WB) detection of cucurbitacin Q1 protein expression levels after administration (n=3). Figure 14 In the figure, A represents the statistical results of p-PI3K protein expression level detection after cucurbitacin Q1 administration; Figure 14 In the figure, B is a statistical graph of the PI3K protein expression level detection results after cucurbitacin Q1 administration; Figure 14 In the figure, C represents the statistical results of p-AKT protein expression level detection after cucurbitacin Q1 administration; Figure 14 D in the figure represents the statistical results of AKT protein expression level detection after cucurbitacin Q1 administration; This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. Detailed Implementation

[0017] This invention provides the use of cucurbitacin Q1 in the preparation of a drug for treating glioma, wherein the chemical structural formula of cucurbitacin Q1 is shown in Formula I. , Formula I.

[0018] In one embodiment, the cucurbitacin Q1 described in this invention has a significant inhibitory effect on glioma cell activity. It can inhibit the protein expression of JAK1, JAK2, and STAT4 by regulating the JAK-STAT signaling pathway, and block the phosphorylation activation of STAT3 protein, thus weakening downstream signal transduction in this pathway. Simultaneously, it can also exert an anti-tumor effect by regulating the phosphorylation level of related proteins in the PI3K-AKT signaling pathway. In another embodiment, the cucurbitacin Q1 used in this invention exhibits potent anti-tumor activity even at low concentrations, with low toxicity to normal nerve cells and good selectivity, providing a novel candidate drug for the clinical treatment of gliomas, and possessing good development value and clinical application prospects.

[0019] In one embodiment, the glioma described in this invention is a grade 1-4 glioma according to the 2021 edition of the WHO Classification of Tumors of the Central Nervous System. In another embodiment, the glioma described in this invention is a grade 4 glioma according to the 2021 edition of the WHO Classification of Tumors of the Central Nervous System. In one embodiment, the glioma described in this invention is a glioma. In one embodiment, the glioma described in this invention is a glioblastoma.

[0020] As one embodiment, the treatment of glioma according to the present invention includes at least one of the following: ① to ④ ① Inhibit the proliferation of glioma cells; ②Inhibit the migration of glioma cells; ③ Inhibit the invasion of glioma cells; ④ Induces apoptosis in glioma cells.

[0021] In one embodiment, the glioma cells described in this invention include human glioma U251 cells. In another embodiment, the cucurbitacin Q1 described in this invention exhibits significant anti-glioma activity even at extremely low concentrations (0.001 μM~25 μM), showing a concentration-dependent inhibitory effect on the proliferation of human glioma U251 cells, effectively reducing tumor cell viability. Furthermore, the cucurbitacin Q1 described in this invention can significantly inhibit the migration and invasion abilities of glioma cells, and the inhibitory effect increases with increasing drug concentration; simultaneously, it can effectively induce glioma cell apoptosis, significantly increasing the tumor cell apoptosis rate.

[0022] In one embodiment, the cucurbitacin Q1 of this invention can inhibit the proliferation of human glioma U251 cells in a concentration-dependent manner, significantly reducing tumor cell viability. In another embodiment, the cucurbitacin Q1 of this invention exhibits a significantly enhanced inhibitory effect on proliferation with increasing concentration within the range of 0.001 μM to 25 μM. In yet another embodiment, after intervention with cucurbitacin Q1 at a concentration of 0.5 μM for 24 hours, the average viability of U251 cells was 26.76%; after intervention with cucurbitacin Q1 at a concentration of 25 μM for 24 hours, the average viability of U251 cells was 19.03%.

[0023] In one embodiment, the cucurbitacin Q1 of this invention can promote the growth of normal human brain astrocytes at low concentrations and inhibit the growth of normal human brain astrocytes at high concentrations. In another embodiment, the cucurbitacin Q1 of this invention has a proliferative effect on the growth of normal human brain astrocytes within a concentration range of 0.001 μM to 0.1 μM. In yet another embodiment, the cucurbitacin Q1 of this invention, at a concentration that effectively inhibits glioma cells (≤25 μM), only produces a mild inhibitory effect on human brain astrocyte SVGP12, and the cell survival rate remains above 70%.

[0024] In one embodiment, the cucurbitacin Q1 of this invention can significantly reduce the migration rate of glioma U251 cells, effectively inhibiting the migration ability of glioma cells, and the inhibitory effect increases with increasing drug concentration and prolonged intervention time. In another embodiment, after intervention with cucurbitacin Q1 at a concentration of 0.05 μM for 36 h, the average migration rate of U251 cells was 19.09%, while the average migration rate of the 0 μM control group at the same time point was 53.04%.

[0025] In one embodiment, the cucurbitacin Q1 of this invention can significantly reduce the number of glioma U251 cells that invasively penetrate the cell membrane, effectively inhibiting the invasive ability of glioma cells, and the inhibitory effect increases with increasing drug concentration. In another embodiment, after intervention with cucurbitacin Q1 at a concentration of 0.05 μM for 24 h, the average number of invasive U251 cells was 228 per field of view, while the average number of invasive cells penetrating the cell membrane in the 0 μM control group was 1028 per field of view.

[0026] In one embodiment, the cucurbitacin Q1 of this invention can significantly increase the apoptosis rate of glioma U251 cells and effectively induce glioma cell apoptosis. In another embodiment, after intervention with cucurbitacin Q1 at a concentration of 1 μM for 24 h, the average apoptosis rate of U251 cells was 32.31%, while the average apoptosis rate of the 0 μM control group was 10.39%.

[0027] In one embodiment, the drug of the present invention comprises a liquid formulation, a solid formulation, or a spray formulation. In one embodiment, cucurbitacin Q1 is the sole active ingredient of the drug. In one embodiment, the drug of the present invention further comprises pharmaceutically acceptable excipients. In one embodiment, the excipients of the present invention comprise one or more of the following: diluents, excipients, fillers, binders, humectants, absorption enhancers, surfactants, lubricants, stabilizers, flavoring agents, sweeteners, and colorings. In one embodiment, the drug of the present invention comprises a liquid formulation, a solid formulation, or a spray formulation.

[0028] In one embodiment, the working concentration of cucurbitacin Q1 in the drug of the present invention is 0.001 μM to 25 μM. In another embodiment, cucurbitacin Q1 of the present invention exhibits inhibitory activity against human glioma U251 cells at working concentrations of 0.001 μM to 25 μM.

[0029] This invention also provides the use of cucurbitacin Q1 in the preparation of products that regulate the JAK-STAT signaling pathway and / or regulate the PI3K-AKT signaling pathway. As one embodiment, the product of this invention includes a drug with therapeutic function for gliomas. As one embodiment, the drug of this invention treats gliomas by regulating the JAK-STAT signaling pathway and / or regulating the PI3K-AKT signaling pathway. As one embodiment, the regulation of the JAK-STAT signaling pathway in this invention includes at least one of the following: 1) Inhibit the expression of at least one of JAK1, JAK2, and STAT4 proteins; 2) Inhibit the phosphorylation activation of STAT3 protein.

[0030] As one implementation method, the regulation of the PI3K-AKT signaling pathway described in this invention includes regulating the phosphorylation level of related proteins (p-PI3K, p-AKT) in the PI3K-AKT signaling pathway.

[0031] In one embodiment, the cucurbitacin Q1 of this invention can inhibit the phosphorylation of STAT3 protein in glioma U251 cells without significantly affecting the expression level of total STAT3 protein. In another embodiment, the cucurbitacin Q1 of this invention can inhibit the protein expression of JAK1, JAK2, and STAT4 in glioma U251 cells, and the inhibitory effect becomes more pronounced with increasing drug concentration. In yet another embodiment, the cucurbitacin Q1 of this invention can inhibit the phosphorylation level of related proteins in the PI3K-AKT signaling pathway in glioma U251 cells.

[0032] To further illustrate the present invention, the application of cucurbitacin Q1 provided by the present invention in the preparation of a drug for treating glioma is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the performance index tests in this embodiment employ conventional testing methods in the art. The terminology used in this invention is merely for describing particular implementation methods and is not intended to limit the scope of the invention. Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains; other raw materials, reagents, test methods, and techniques not specifically mentioned in this invention refer to raw materials and reagents commonly used by one of ordinary skill in the art, as well as commonly employed experimental methods and techniques.

[0034] The cucurbitacin Q1 used in the following examples is named E,6R)-6-hydroxy-2-methyl-5-oxo-6-[(2S,3S,8S,9R,10R,13R,14S,16R,17R)-2,3,16-trihydroxy-4,4,9,13,14-pentamethyl-11-oxo-1,2,3,7,8,10,12,15,16,17-decahydro-cyclopentanol-17-yl]-hept-3-en-2-yl]acetate, a natural compound extracted from the stem of a melon; CAS Registry Number: 99530-82-2; Chemical Formula: C 32 H 48 O8; molecular weight 560.72; white to grayish-white fixed appearance; soluble in ethanol, DMSO and other organic solvents, but with limited solubility in water, classifying it as a drug with low water solubility.

[0035] In the following examples, cucurbitacin Q1 was purchased from Solarbio, catalog number: IYT2019, CAS number: 99530-82-2.

[0036] Example 1 Effect of cucurbitacin Q1 on the proliferation of human glioma U251 cells (CCK-8 assay) This study investigated the effects of different concentrations of cucurbitacin Q1 on the proliferation of human glioma cells U251.

[0037] 1. Test Methods Resuspend healthy human glioma cells (U251 cells) at a ratio of 7 × 10⁻⁶. 3Cells were seeded per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was carefully aspirated, and 0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 25 μM cucurbitacin Q1 were added to each well, with three parallel wells per group. The cells were incubated for another 24 hours, and then 10 μL of LCK-8 solution (protected from light) was added to each well. The cells were incubated at 37°C for 1 hour. The absorbance (OD value) of each well was then measured using a microplate reader at 450 nm. The OD value of the 0 μM group was normalized to 100%, and cell viability was calculated based on the absorbance values ​​using the formula shown in Equation I. Cell viability = (OD value of drug-treated group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%, Formula I.

[0038] 2. Experimental Results and Conclusions The viability of U251 cells obtained after 24 hours of intervention with different concentrations of cucurbitacin Q1 is shown in Table 1. Figure 1 As shown in the results, the proliferation rate of U251 cells was significantly slowed down after cucurbitacin Q1 intervention, indicating that cucurbitacin Q1 showed a significant inhibitory effect on the cell proliferation rate of tumor cells, and that cucurbitacin Q1 has a significant anti-tumor effect.

[0039] Table 1. Cell viability assay results of U251 cells after proliferation culture under different concentrations of cucurbitacin Q1 intervention.

[0040] Example 2 Effects of cucurbitacin Q1 on the proliferation of normal human brain astrocytes SVGP12 (CCK-8 assay) This study investigated the effects of different concentrations of cucurbitacin Q1 on the proliferation of normal human brain astrocytes SVGP12.

[0041] 1. Test Methods Resuspend healthy human brain SVGP12 astrocytes at a ratio of 5 × 10⁻⁶. 3Cells were seeded per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was carefully aspirated, and 0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 25 μM cucurbitacin Q1 were added to each well, with three parallel wells per group. The cells were incubated for another 24 hours, and then 10 μL of LCK-8 solution (protected from light) was added to each well. The cells were incubated at 37°C for 1 hour. The absorbance (OD value) of each well was then measured using a microplate reader at 450 nm. The OD value of the 0 μM group was normalized to 100%, and cell viability was calculated based on the absorbance values ​​using the formula shown in Equation I. Cell viability = (OD value of drug-treated group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%, Formula I.

[0042] 2. Experimental Results and Conclusions The viability of SVGP12 cells obtained after 24 hours of intervention with different concentrations of cucurbitacin Q1 is shown in Table 2. Figure 2 As shown in the results, the cell viability of SVGP12 cells initially increased and then decreased after cucurbitacin Q1 intervention. At concentrations of 0.001 μM to 0.01 μM, cell viability was slightly higher than the control group (SVGP12 cells with 0 μM drug); at concentrations of 0.5 μM and above, cell viability decreased in a concentration-dependent manner. These results indicate that cucurbitacin Q1, within the concentration range (≤25 μM) that effectively inhibits the proliferation of glioma U251 cells, only exhibits a mild inhibitory effect on normal human brain astrocytes SVGP12, with cell survival remaining above 70%, demonstrating good safety and tumor selectivity.

[0043] Table 2. Cell viability assay results of SVGP12 cells after proliferation culture under different concentrations of cucurbitacin Q1 intervention.

[0044] Example 3 Effects of cucurbitacin Q1 on the migration of human glioma U251 cells (cell scratch assay) The cell scratch assay is a common method used in the laboratory to analyze cell migration ability. This example studies the effects of different concentrations of cucurbitacin Q1 on the cell migration of human glioma U251 cells.

[0045] 1. Test Methods First, using a marker pen, draw evenly spaced horizontal lines on the back of the 6-well plate, approximately every 0.5–1 cm, passing through each well. Ensure at least 5 lines pass through each well. Resuspend healthy U251 human glioma cells and seed them into each well at a rate of 2 mL per well (2 × 10⁶ cells). 5 Cells per well were incubated at 37°C with 5% CO2. After 24 hours, when the confluence of cells in each well was approximately 80%, a 200µl pipette tip was used at a 45° angle to the cell plane to make a scratch on the cell layer along the line drawn on the back of the plate on the first day (it is best to use the same pipette tip for different wells). The culture medium was carefully aspirated, and the cells were washed twice with PBS. Since the scratch assay requires a cell concentration that does not significantly affect cell proliferation, 0μM, 0.001μM, 0.01μM, and 0.05μM concentrations of cucurbitacin Q1 were added to different wells, with three parallel wells in each group. Microscopic images were taken at 0h, 12h, 24h, and 36h, and the migration rate was calculated.

[0046] 2. Experimental Results and Conclusions Table 3 shows the migration rate of U251 cells at different time points after intervention with different concentrations of cucurbitacin Q1. Figure 3 and Figure 4 As shown in the results, compared with the control group without cucurbitacin Q1, the migration rate of glioma cells U251 was significantly reduced after administration of cucurbitacin Q1. P The concentration of cucurbitacin Q1 (<0.05) indicates that cucurbitacin Q1 can significantly inhibit the migration ability of glioma cells and even glioblastoma cells, and the inhibitory effect increases with increasing drug concentration.

[0047] Table 3. Results of U251 cell migration rate after cucurbitacin Q1 intervention.

[0048] Example 4 Effects of cucurbitacin Q1 on the migration of human glioma U251 cells (Transwell migration assay) This study investigated the effects of different concentrations of cucurbitacin Q1 on the Transwell migration of human glioma U251 cells.

[0049] 1. Test Methods Human glioma cells (U251) in good growth condition were starved 12 hours in advance, then digested and resuspended, and seeded into 24-well upper chambers (5×10⁶ cells per well) with 100 μL of medium containing cucurbitacin Q1 (0 μM, 0.001 μM, 0.01 μM, 0.05 μM) (1% serum concentration). 4Add 500 μL of complete culture medium (10% serum concentration) to the lower chamber of each well and incubate at 37°C with 5% CO2. There are 3 parallel wells per group. After 24 h of incubation, remove the cells, discard the waste liquid, add 1 mL of 4% paraformaldehyde for fixation for 15 min, add 500 μL of 0.1% crystal violet to each well for staining for 15 min, then gently wipe away the upper layer of cells with a cotton swab, wash twice with PBS, and air dry. After air drying, observe and photograph under a 10X microscope.

[0050] 2. Experimental Results and Conclusions The Transwell cell number detection results of U251 cells after intervention with different concentrations of cucurbitacin Q1 for 24 hours are shown in Table 4. Figure 5 and Figure 6 As shown in the results, compared with the control group without cucurbitacin Q1, the number of migrating U251 glioma cells was significantly reduced after administration of cucurbitacin Q1. P Cucurbitacin Q1 (<0.05) inhibited the transwell migration of human glioma cells U251.

[0051] Table 4. Transwell cell migration results of U251 cells after cucurbitacin Q1 intervention (cells / field)

[0052] Example 5 Effects of cucurbitacin Q1 on the invasion of human glioma U251 cells (Transwell invasion assay) This study investigated the effects of different concentrations of cucurbitacin Q1 on the Transwell invasion ability of human glioma U251 cells.

[0053] 1. Test Methods First, healthy human glioma cells (U251) were starved 12 hours in advance. Then, a 1:7 mixture of Matting gel (Corning, 35620) and culture medium was added to each well, and the cells were incubated at 37°C for 8 hours until solidification. The cells were then digested and resuspended, and seeded in 24-well upper chambers (8×10⁶ cells per well) with 100 μL of cucurbitacin Q1 (0 μM, 0.001 μM, 0.01 μM, 0.05 μM) medium (1% serum concentration). 4Add 500 μL of complete culture medium (10% serum content) to the lower chamber of each well and incubate at 37°C with 5% CO2. There are 3 parallel wells per group. After 24 h of incubation, remove the cells, discard the waste liquid, add 1 mL of 4% paraformaldehyde for fixation for 15 min, add 500 μL of 0.1% crystal violet to each well for staining for 15 min, then gently wipe away the upper layer of cells with a cotton swab, wash twice with PBS, and air dry. After air drying, observe and photograph under a 10X microscope.

[0054] 2. Experimental Results and Conclusions The Transwell cell count of U251 cells after 24 hours of intervention with different concentrations of cucurbitacin Q1 is shown in Table 5. Figure 7 and Figure 8 As shown in the results, compared with the control group without cucurbitacin Q1, the number of U251 cells gradually decreased with increasing drug concentration. Therefore, our experimental results indicate that cucurbitacin Q1 inhibited the transwell invasion of human glioma cells U251.

[0055] Table 5. Detection results of cucurbitacin Q1 intervention in Transwell invasion of U251 (number of cells / field of view)

[0056] Example 6 Effect of cucurbitacin Q1 on apoptosis in human glioma U251 cells (flow cytometry) This embodiment investigated the effect of cucurbitacin Q1 intervention on apoptosis in human glioma U251 cells.

[0057] 1. Test Methods Resuspend healthy human glioma cells (U251 cells) at a ratio of 3 × 10⁻⁶. 5 Each sample was seeded into a 6-well plate and incubated at 37°C with 5% CO2 for 24 h. After carefully aspirating the culture medium and washing twice with PBS, 0 μM, 0.1 μM, 0.5 μM, and 1 μM concentrations of cucurbitacin Q1 were added to different wells, with 3 parallel wells per group. The plates were then incubated for another 24 h. After resuspending the plates, trypsin was added for digestion, and the plates were washed twice with PBS. Then, 5 μL of Annexin V-FITC and 5 μL of PI dye were added, and the plates were incubated at room temperature for 5-15 min. The samples were then analyzed by flow cytometry.

[0058] 2. Experimental Results and Conclusions The apoptosis rate of U251 cells after intervention with different concentrations of cucurbitacin Q1 for 24 hours is shown in Table 6. Figure 9 and Figure 10As shown in the results, compared with the control group without cucurbitacin Q1, the apoptosis rate of glioma cells U251 was significantly increased after administration of cucurbitacin Q1. P<0.05, (P<0.01) Cucurbitacin Q1 promoted the apoptosis ability of glioma cells.

[0059] Table 6. Results of Detection of Apoptosis Rate in Human Glioma Cells U251 under Cucurbitacin Q1 Intervention

[0060] Example 7 Effects of cucurbitacin Q1 on JAK-STAT pathway-related proteins in human glioma U251 cells (Western Blot assay) This embodiment investigated the effect of cucurbitacin Q1 intervention on apoptosis in human glioma U251 cells.

[0061] 1. Test Methods Resuspend healthy human glioma cells (U251 cells) at a ratio of 3 × 10⁻⁶. 5Inoculate each well with 1 sample of cucurbitacin Q1 into a 6-well plate and incubate at 37°C with 5% CO2 for 24 h. Carefully aspirate the culture medium, wash twice with PBS, and add 0 μM, 0.1 μM, 0.5 μM, and 1 μM concentrations of cucurbitacin Q1 to different wells, with 3 parallel wells per group. Incubate for another 24 h, then add RIPA lysis buffer to extract protein. Centrifuge at 12000 rpm for 10 min, and determine protein concentration using BCA. Based on the calculated concentration, add SDS for denaturation. After denaturation, allow the samples to cool slowly. For each group of samples, use 50 μg of protein per well and run in a precast gel at 110 V constant voltage for 85 min. Transfer the PVDF membrane at 400 mA for 45 min, and then seal the membrane with a rapid sealing agent. After blocking with blocking solution (Yesen, 36122ES76) for 10 min, the corresponding primary antibodies [JAK1 (Proteintech, 66466-1-Ig), JAK2 (CST, D2E12), p-STAT3 (CST, 9145), STAT3 (CST, 12640), STAT4 (CST, 2653), β-actin (Proteintech, 66009-1-Ig, all diluted 1:1000)] were incubated overnight. After overnight incubation, the membrane was washed 4 times with TBST, and the secondary antibodies [HRP-conjugated Goat Anti-Rabbit IgG (H+L) (Proteintech, SA00001-2), HRP-conjugated Goat Anti-Mouse IgG (H+L) (Proteintech, SA00001-1, all diluted 1:2000)] were added. The membrane was incubated at room temperature for 1 h, washed 4 times with TBST, and then developed.

[0062] 2. Experimental Results and Conclusions After 24 hours of intervention with different concentrations of cucurbitacin Q1, the expression results of JAK-STAT signaling pathway-related proteins in U251 cells were obtained as follows: Figure 11 and Figure 12As shown in the results, cucurbitacin Q1 inhibited the expression of phosphorylated STAT3 protein, but did not affect the expression of non-phosphorylated STAT3. Furthermore, the experiment showed that cucurbitacin Q1 inhibited the expression of non-phosphorylated JAK1, non-phosphorylated JAK2, and non-phosphorylated STAT4 proteins, with the inhibitory effect becoming more pronounced with increasing drug concentration. This indicates that cucurbitacin Q1, through transcriptional or post-translational regulatory mechanisms, directly reduces the protein synthesis or stability of upstream kinases (JAK1 / JAK2) and downstream transcription factors (STAT4) in the JAK-STAT pathway, weakening the signal transduction capacity of this pathway at its source. In addition to reducing the total amount of JAK protein, cucurbitacin Q1 further inhibited the tyrosine phosphorylation activation process of STAT3. Since the transcriptional activity of STAT3 is strictly dependent on its phosphorylation, this effect directly blocks the core signal output downstream of the JAK-STAT pathway. These experimental results indicate that cucurbitacin Q1 inhibits U251 by regulating the JAK-STAT signaling pathway, thereby exerting an anti-tumor effect.

[0063] Example 8 Effects of cucurbitacin Q1 on the expression of PI3K-AKT signaling pathway-related proteins in human glioma U251 cells (Western Blot method) This embodiment investigated the effect of cucurbitacin Q1 intervention on apoptosis in human glioma U251 cells.

[0064] 1. Test Methods Resuspend healthy human glioma cells (U251 cells) at a ratio of 3 × 10⁻⁶. 5Each sample was seeded into a 6-well plate and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was carefully aspirated, and the plates were washed twice with PBS. Cucurbitacin Q1 at concentrations of 0 μM, 0.1 μM, 0.5 μM, and 1 μM was added to different wells, with three parallel wells per group. The plates were incubated for another 24 hours. RIPA lysis buffer was then added to extract the protein. After centrifugation at 12000 rpm for 10 min, protein concentration was determined using BCA. Based on calculations, the protein sample weight per well was 50 μg. SDS was added for denaturation. The denatured samples were slowly cooled and run in a pre-cast gel at a constant voltage of 110 V for 85 min, followed by a 400 m... After transfer for 45 min, the PVDF membrane was blocked with YESEN rapid blocking buffer for 10 min, followed by incubation with primary antibodies [p-PI3K (CST, 4228), PI3K (CST, 4257), p-AKT (HuaBio, ET1607-73), AKT (CST, 9272), β-actin (Proteintech, 66009-1-Ig, all diluted 1:1000], overnight. After overnight incubation, the membrane was washed four times with TBST, and secondary antibodies [HRP-conjugated Goat Anti-Rabbit IgG (H+L) (Proteintech, SA00001-2, all diluted 1:2000)] were added. After washing four times with TBST, the membrane was developed. The results are as follows. Figure 13 and Figure 14 As shown.

[0065] 2. Experimental Results and Conclusions Depend on Figure 13 and Figure 14 The results clearly show that cucurbitacin Q1 may exert its anti-tumor effect by regulating the PI3K / AKT signaling pathway and affecting the phosphorylation level of related proteins.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of cucurbitacin Q1 in the preparation of drugs for treating gliomas, characterized in that, The chemical structural formula of cucurbitacin Q1 is shown in Formula I. Equation I.

2. The application according to claim 1, characterized in that, The glioma in question is a brain glioma.

3. The application according to claim 2, characterized in that, The glioma is a glioblastoma.

4. The application according to claim 1, characterized in that, The treatment of glioma includes at least one of the following: ① to ④ ① Inhibit the proliferation of glioma cells; ②Inhibit the migration of glioma cells; ③ Inhibit the invasion of glioma cells; ④ Induces apoptosis in glioma cells.

5. The application according to claim 1, characterized in that, The drug includes liquid formulations, solid formulations, or spray formulations.

6. The application according to claim 1, characterized in that, Cucurbitacin Q1 is the sole active ingredient of the drug.

7. The application according to claim 1, characterized in that, The working concentration of cucurbitacin Q1 in the drug is 0.001 μM to 25 μM.

8. Application of cucurbitacin Q1 in the preparation of products that regulate the JAK-STAT signaling pathway and / or regulate the PI3K-AKT signaling pathway.

9. The application according to claim 8, characterized in that, The regulation of the JAK-STAT signaling pathway includes at least one of the following: 1) Inhibit the expression of at least one of JAK1, JAK2, and STAT4 proteins; 2) Inhibit the phosphorylation activation of STAT3 protein.

10. The application according to claim 8, characterized in that, The regulation of the PI3K-AKT signaling pathway includes regulating the phosphorylation level of related proteins in the PI3K-AKT signaling pathway.

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