Application of jujuboside A in treatment of bladder cancer

Abstract

The invention discloses application of jujuboside A in treatment of bladder cancer. Cell experiments prove that jujuboside A can reduce the activity of bladder cancer cells and induce apoptosis of the bladder cancer cells, and can inhibit expression of ATP1A2 in the bladder cancer cells and mitochondrial energy metabolism. The application provides a brand new thought for research and development of bladder cancer treatment medicines, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of spina date seed saponin A in treatment of bladder cancer. BACKGROUND

[0002] Bladder cancer is a common malignant tumor of the urinary system. Due to its high recurrence, drug treatment, especially chemical drug treatment, is particularly important in the treatment of bladder cancer. The chemical drug treatment of bladder cancer mainly includes bladder perfusion chemotherapy and systemic chemotherapy. Bladder perfusion chemotherapy is mainly used for postoperative intravesical perfusion treatment of non-muscular invasive bladder cancer to prevent tumor recurrence, but due to the heterogeneity of tumors, a certain proportion of tumors are not sensitive to drugs, resulting in tumor recurrence.

[0003] Therefore, it is necessary to develop and use new drugs capable of treating bladder cancer. SUMMARY

[0004] In order to make up for the deficiency of the prior art, the application provides application of spina date seed saponin A in treatment of bladder cancer.

[0005] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme: The first aspect of the application provides application of spina date seed saponin A in preparation of a drug for treating bladder cancer.

[0006] Further, the drug further comprises other drugs for treating bladder cancer.

[0007] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0008] Further, the pharmaceutically acceptable excipient comprises an excipient, a buffer, a surfactant, a preservative.

[0009] Further, the dosage form of the drug comprises a gastrointestinal administration dosage form and a non-gastrointestinal administration dosage form.

[0010] Further, the gastrointestinal administration dosage form comprises tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent preparations, emulsions, suspensions, syrup, drops, chewable preparations.

[0011] Further, the non-gastrointestinal administration dosage form comprises an injection administration dosage form, a respiratory administration dosage form, a cavity administration dosage form, a mucosal administration dosage form, and a skin administration dosage form.

[0012] The second aspect of the application provides a drug for treating bladder cancer, wherein the drug comprises spina date seed saponin A.

[0013] The third aspect of the present application provides a method for inhibiting bladder cancer cell viability / apoptosis in vitro, the method comprising administering Jujuboside A.

[0014] Further, the bladder cancer cell is a T24 cell.

[0015] Further, the method is a method for non-therapeutic purposes.

[0016] The fourth aspect of the present application provides a method for preparing a cell with reduced ATP1A2 level, the method comprising administering Jujuboside A.

[0017] Further, the cell is a bladder cancer cell.

[0018] Further, the bladder cancer cell is a T24 cell.

[0019] The fifth aspect of the present application provides use of Jujuboside A in the preparation of a product for regulating Bcl-2 level, mitochondrial energy metabolism related protein level in a cell.

[0020] Further, the mitochondrial energy metabolism related protein comprises any one or more of ATP1A2, ATP5A, COX1, COX2, NDUFA1, UQCRC2, MTCO1, SDHB and NDUFB8.

[0021] Further, the cell is a bladder cancer cell.

[0022] Further, the bladder cancer cell is a T24 cell.

[0023] Further, the method is a method for non-therapeutic purposes.

[0024] The sixth aspect of the present application provides use of Jujuboside A in the preparation of a product for inhibiting mitochondrial energy metabolism.

[0025] Further, the inhibition of mitochondrial energy metabolism comprises any one or more of reduction of mitochondrial membrane potential, ATP content, oxygen consumption rate, extracellular acidification rate.

[0026] Further, the inhibition of mitochondrial energy metabolism is inhibition of mitochondrial energy metabolism in a bladder cancer cell. Further, the bladder cancer cell is a T24 cell.

[0027] Advantages and beneficial effects of the present application: The present application proves by cell experiments that Jujuboside A can reduce the viability of bladder cancer cells and induce apoptosis, can inhibit the expression of ATP1A2 and mitochondrial energy metabolism in bladder cancer cells. The present application provides a brand-new idea for the research and development of bladder cancer treatment drugs, and has broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 FIG. 1 is a JuA target gene screening diagram, wherein 1A is a principal component analysis (PCA) diagram of the GSE133624 dataset, 1B is a volcano plot of DEGs in the GSE133624 dataset, 1C is a cluster heat map of DEGs in the GSE133624 dataset, 1D is a gene ontology (GO) function enrichment analysis diagram of DEGs, 1E is a Kyoto Encyclopedia of Genes and Genomes (KEGG) function enrichment analysis diagram of DEGs, 1F is a Venn diagram of the overlap of DEGs in the GSE133624 dataset and predicted JuA target genes, 1G is a protein-protein interaction (PPI) analysis diagram of IL-6, ATP1A2 and HSD11B1, and 1H is a prognosis analysis diagram of IL-6, ATP1A2 and HSD11B1 in the Cancer Genome Atlas (TCGA) bladder cancer cohort; Figure 2 FIG. 2 is a diagram showing that ATP1A2 overexpression promotes bladder cancer cell proliferation and inhibits apoptosis, wherein 2A is a diagram showing the relative expression level of ATP1A2 protein in normal human prostate epithelial cells and different bladder cancer cell lines, 2B is a diagram showing the relative expression level of ATP1A2 mRNA in normal human prostate epithelial cells and different bladder cancer cell lines, 2C is a diagram showing the relative expression level of ATP1A2 mRNA after transfection of ATP1A2 overexpression plasmid into 5637 cells, 2D is a diagram showing the viability of 5637 cells after transfection of ATP1A2 overexpression plasmid vector, 2E is a diagram showing the apoptosis rate of 5637 cells after transfection of ATP1A2 overexpression plasmid vector, and 2F is a diagram showing the relative expression level of Bax, Bcl-2, caspase-3 and cleaved-caspase-3; Figure 3Figure 3 is an analysis diagram of ATP1A2 overexpression enhancing the energy metabolism of bladder cancer cells, wherein 3A is a mitochondrial membrane potential diagram of 5637 cells after transfection of the ATP1A2 overexpression plasmid vector (x400; scale: 25 μm), 3B is a diagram of the change in ATP level in 5637 cells after transfection, 3C is a diagram of the change in ROS level in 5637 cells after transfection, 3D is a diagram of the morphological change of mitochondrial ultrastructure in 5637 cells after transfection (black arrow, normal mitochondria; red arrow, damaged mitochondria; N, nucleus; M, mitochondria; x3000, scale: 5 μm; x8000, scale: 2 μm; x20000, scale: 500 nm), 3E is an OCR and ECAR diagram of 5637 cells after ATP1A2 overexpression, 3F is a diagram of the change in expression level of mitochondrial energy metabolism related proteins (ATP1A2, ATP5A, COX1, COX2 and NDUFA1), and 3G is a diagram of the change in expression level of mitochondrial energy metabolism related proteins (UQCRC2, MTCO1, SDHB, NDUFB8) detected by Western blotting; Figure 4 Figure 4 is an analysis diagram of ATP1A2 knockdown inhibiting the proliferation of bladder cancer cells and inducing apoptosis, wherein 4A is a diagram of the relative expression level of ATP1A2 mRNA after transfection of siATP1A2 into T24 cells, 4B is a diagram of the change in cell viability after transfection of siRNA into T24 cells, 4C is a diagram of the change in apoptosis rate of T24 cells after transfection of siRNA, 4D is a histogram of statistical analysis of the apoptosis rate, and 4E is a diagram of the relative expression level of apoptosis related proteins Bax, Bcl-2, caspase-3 and cleaved-caspase-3; Figure 5 Figure 5 is an analysis diagram of ATP1A2 knockdown inhibiting the energy metabolism of bladder cancer cells, wherein 5A is a diagram of the change in mitochondrial membrane potential in T24 cells after transfection of siRNA (x400; scale: 25 μm), 5B is a diagram of the change in ATP level in T24 cells after transfection, 5C is a diagram of the change in ROS level in T24 cells after transfection, 5D is a diagram of the morphological change of mitochondrial ultrastructure in T24 cells after transfection (black arrow, normal mitochondria; red arrow, damaged mitochondria; N, nucleus; M, mitochondria; x3000, scale: 5 μm; x8000, scale: 2 μm; x20000, scale: 500 nm), 5E is an OCR and ECAR determination diagram of T24 cells after ATP1A2 knockdown, 5F is a diagram of the expression level of mitochondrial energy metabolism related proteins (ATP1A2, ATP5A, COX1, COX2 and NDUFA1), and 5G is a diagram of the change in expression level of mitochondrial energy metabolism related proteins (UQCRC2, MTCO1, SDHB, NDUFB8) detected by Western blotting; Figure 6 This is an analysis of JuA's inhibition of ATP1A2-induced apoptosis in bladder cancer cells. 6A shows the two-dimensional (2D) and three-dimensional (3D) chemical structures of JuA and their relative molecular masses; 6B shows the molecular docking diagram of JuA with ATP1A2 protein (proteins are represented by blue cartoons, and compounds by cyan bars); 6C shows the changes in the relative expression level of ATP1A2 protein in T24 cells after treatment with different concentrations of JuA; 6D shows the changes in cell viability of T24 cells after treatment with different concentrations of JuA; 6E shows the changes in cell viability of T24 cells after treatment with JuA and transfection with an ATP1A2 overexpression plasmid; 6F shows the changes in the apoptosis rate of T24 cells after treatment with JuA and transfection with an ATP1A2 overexpression plasmid; and 6G shows the relative expression levels of apoptosis-related proteins Bax, Bcl-2, caspase-3, and cleaved-caspase-3 in T24 cells after treatment with JuA and transfection with an ATP1A2 overexpression plasmid. Figure 7 This is an analysis of the mitochondrial energy metabolism disorder mediated by JuA in bladder cancer cells. 7A shows the change in mitochondrial membrane potential in T24 cells after JuA treatment and transfection with an ATP1A2 overexpression plasmid; 7B shows the change in intracellular ATP levels in T24 cells after JuA treatment and transfection with an ATP1A2 overexpression plasmid; 7C shows the change in intracellular ROS levels in T24 cells after JuA treatment and transfection with an ATP1A2 overexpression plasmid; 7D shows the morphological changes in the ultrastructure of mitochondria in T24 cells after JuA treatment and transfection with an ATP1A2 overexpression plasmid (black arrow: normal mitochondria; red arrow: damaged mitochondria; N: nucleus; M: mitochondria; ×3000, scale bar: 5 μm; ×8000, scale bar: 2 μm; ×20000, scale bar: 500). 7E shows the OCR and ECAR images of T24 cells after treatment with JuA and transfection with the ATP1A2 overexpression plasmid. 7F shows the expression changes of mitochondrial energy metabolism-related proteins (ATP1A2, ATP5A, COX1, COX2, and NDUFA1) in T24 cells after treatment with JuA and transfection with the ATP1A2 overexpression plasmid. 7G shows the expression level changes of mitochondrial energy metabolism-related proteins (UQCRC2, MTCO1, SDHB, and NDUFB8).

[0029] In the figure, n=6, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, P>0.05. Detailed Implementation

[0030] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] This invention provides the application of jujube seed saponin A in the preparation of a drug for treating bladder cancer.

[0032] In some embodiments, jujuboside A, also known as Jujuboside A or JuA, is the dried, mature seed of Ziziphus jujuba Mill. var. spinosa (Bunge) Huex H.F. Chou, a plant belonging to the genus Ziziphus in the family Rhamnaceae. It is also known as jujube kernel or sour jujube seed. It has a sweet taste and neutral properties, and has the effects of nourishing the liver, calming the mind, astringing sweat, and promoting body fluid production. It is used for symptoms such as insomnia due to deficiency and restlessness, palpitations and excessive dreaming, excessive sweating due to physical weakness, and thirst due to fluid depletion. The Compendium of Materia Medica lists it as a superior medicine.

[0033] In some implementations, treatment refers to the medical management of a patient aimed at curing, improving, stabilizing, or preventing a disease, pathological state, or symptom. Treatment includes active therapy, which is treatment specifically aimed at improving a disease, pathological state, or symptom, and also includes etiological treatment, which is treatment aimed at eliminating the cause of the relevant disease, pathological state, or symptom. Furthermore, treatment includes palliative care, which is treatment designed to relieve symptoms rather than cure a disease, pathological state, or symptom; preventive care, which is treatment aimed at minimizing or partially or completely suppressing the development of a relevant disease, pathological state, or symptom; and supportive care, which is treatment used to supplement another specific therapy aimed at improving a relevant disease, pathological state, or symptom. Specifically, treatment refers to various indicators of maintaining and / or administering medication to prevent and reduce the occurrence or development of a disease, thereby inhibiting, suppressing, alleviating, improving, slowing, stopping, delaying, or reversing the progression or exacerbation of the disease. These indicators include the reduction or elimination of symptoms or complications, or the cure or elimination of the disease, disorder, or condition.

[0034] The medication also includes other drugs for treating bladder cancer.

[0035] In some implementations, other drugs for treating bladder cancer include, but are not limited to, chemotherapy drugs, immunotherapy drugs, and targeted drugs.

[0036] The drug also includes pharmaceutically acceptable excipients.

[0037] In some embodiments, pharmaceutically acceptable excipients include all solvents, diluents, buffers (e.g., neutral buffered saline, or optionally Tris-HCl, acetate, or phosphate buffers), solubilizers (e.g., polysorbate 80), colloids, dispersion media, solvents, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavoring agents, aroma enhancers, thickeners, substances that provide storage effects, coating agents, antifungal agents, preservatives (e.g., Thimerosal, benzyl alcohol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, extenders (e.g., lactose, mannitol), etc. The use of such media and reagents for formulating pharmaceuticals is well known in the art. Their use in pharmaceuticals may be considered unless any conventional media or reagent is incompatible with the active ingredient.

[0038] The dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0039] In some embodiments, the gastrointestinal dosage forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release formulations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0040] In some embodiments, the non-gastrointestinal dosage form includes injectable dosage form, respiratory dosage form, cavity dosage form, mucosal dosage form, and skin dosage form.

[0041] In some embodiments, the injectable dosage forms include, but are not limited to, various injectables such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections; the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder inhalers; the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills, for use in the rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ointments, mouthwashes, sublingual tablets, adhesive tablets, and patches; and the skin dosage forms include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0042] In some embodiments, the dosage of the drug described in this application depends on several factors, including: the method of administration, the severity of the disease, the treatment or prevention of cancer, and the age, weight, and health status of the subject to be treated.

[0043] In some embodiments, the administration of the drug is not limited to a specific pattern of administration, dosage, or frequency. The drug may be administered to the subject in a single dose or in multiple doses. For example, the drug may be administered once a week, or once every 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is understood that for any given subject, the specific dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the drug.

[0044] In some embodiments, any of these strategies can be pursued to achieve controlled release, wherein the release rate exceeds the metabolic rate of the drug under consideration. In some embodiments, controlled release is achieved through a variety of dosage form parameters and components, including, for example, appropriate selection of various types of controlled-release drugs and coatings. Examples include single or multi-unit tablets or capsules, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.

[0045] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0046] Example 1. Materials and Methods 1) Data grouping This study used the Gene Expression Comprehensive Database (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ) to obtain expression data. The GSE133624 dataset (transcriptomic analysis of gene expression levels in bladder urothelial carcinoma and adjacent normal tissues) was employed to investigate differential expression between the bladder cancer group (case group; bladder cancer tissue) and the control group (Ctrl group; adjacent normal tissue). In the GSE133624 dataset, the sample ratio of the case group to the control group was 36:29.

[0047] 2) Differential mRNA expression analysis using the GSE133624 dataset. Differential expression between the case and control groups was analyzed using the "limma" package. The selection criteria were |log2FC|>1 and P<0.05. A total of 1845 differentially expressed genes (DEGs) were identified, of which 451 were upregulated and 1394 were downregulated. Based on the detected differentially expressed genes, principal component analysis was performed on both groups using the R packages "FactoMineR" and "factoextra". Cluster heatmaps of differentially expressed genes were plotted using the R package "pheatmap", and the significance of log2FC values ​​and differentially expressed genes was visualized using the R package "ggplot2".

[0048] 3) Perform GO and KEGG functional enrichment analysis on differentially expressed genes in the GSE133624 dataset. The R package “clusterProfiler” was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses on DEGs obtained from the GSE133624 dataset to identify relevant GO functions and KEGG pathways, and the top-ranked results were recorded.

[0049] 4) Screening of key JuA target genes in bladder cancer Using "jujube seed saponin A" as the keyword, target genes of jujube seed saponin A were searched in the Encyclopedia of Traditional Chinese Medicine database (http: / / www.tcmip.cn / ETCM / index.php / Home / Index / index.html). Overlap analysis was performed between the target genes of jujube seed saponin A and identified differentially expressed genes. Venn diagrams were constructed using the "Venn Diagram" package in R. A protein-protein interaction (PPI) network of overlapping genes was constructed using the GENEMANIA database (http: / / genemania.org / ). Kaplan-Meier survival analysis was performed using survival data from the TCGA bladder urothelial carcinoma cohort to assess the correlation between the expression levels of target genes (IL-6, ATP1A2, and HSD11B1) and overall survival (OS). The significance threshold was set at P < 0.05.

[0050] 5) Molecular docking The structures of the active compounds were downloaded from the PubChem database and imported into ChemBio3D 14.0 software to adjust the spatial conformation of the active ingredients and calculate energy optimization. The three-dimensional crystal structure of the target protein was downloaded from the Uniprot database. Water molecules and organic matter in the target protein were removed using PyMOL, and then the target protein was imported into AutoDockTools 1.5.6 for hydrogenation, charge distribution, and atom type addition. Molecular docking was performed using AutoDock Vina, and the docking results were plotted using DiscoveryStudio 2021 and PyMOL 2.6.1.

[0051] 6) Cell Culture The SV-HUC-1 normal human bladder epithelial cell line (CL-0222) and the UMUC3 (CL-0463), 5637 (CL-0002), RT4 (CL-0431), T24 (CL-0227), and J82 (CL-0125) human bladder cancer cell lines were all purchased from Wuhan Pricella Biotechnology Co., Ltd., and were confirmed to be free of mycoplasma contamination by short tandem repeat (STR) typing. All cells were cultured in high-glucose Dulbecco modified Eagle medium (DMEM; PM150210B; Wuhan Pricella Biotechnology Co.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were placed in a cell culture incubator (120300; Thermo Fisher Scientific, China) and cultured at 37°C and 5% CO2. The medium was changed every other day. After reaching the exponential growth phase, the cells were used for subsequent experiments.

[0052] 7) Cell transfection and grouping Next, 5637 and T24 cells in the exponential growth phase were collected. 2 mL of trypsin was added, and the cells were incubated for 3 minutes. Then, 3 mL of high-glucose DMEM medium was added, and the cell suspension was collected in centrifuge tubes and centrifuged at 1200×g for 3 minutes. After adding 3 mL of high-glucose DMEM medium, cell density was calculated using a cell counter (A49866; Thermo Fisher Scientific). Cells were then cultured at 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. Specific interfering RNA and overexpression plasmids were designed for ATP1A2. The following day, following the transfection reagent manufacturer's instructions (11668500; Thermo Fisher Scientific), a mixture of the overexpression plasmid and transfection reagent was added to 5637 cells, while a mixture of the interfering RNA and transfection reagent was added to T24 cells. After 4 hours of cell culture, the medium was changed, and cells were cultured for another 48 hours. RT-qPCR and Western blotting analyses were used to verify the construction of the ATP1A2 overexpression and knockdown cell models.

[0053] 8) Cell grouping to verify the effect of high and low ATP1A2 levels on bladder cancer progression. 5637 cells were divided into two groups: an overexpression control group (vector) and an ATP1A2 overexpression group (oeATP1A2). T24 cells were divided into three groups: a knockdown control group (siNC), an ATP1A2 first knockdown group (siATP1A2-1), and an ATP1A2 second knockdown group (siATP1A2-2) (Table 1). To verify the pharmacological effects of JuA (HY-N0659; MedChemExpress, China), bladder cancer cells were treated with different concentrations of JuA and divided into four groups: control group, JuA-L group (12.5 μmol / L), JuA-M group (25 μmol / L), and JuA-H group (50 μmol / L). To determine whether the pharmacological effects of JuA depended on ATP1A2, cells were divided into three groups: control group, JuA-H (50 μmol / L) group, and JuA-H (50 μmol / L) + oeATP1A2 group.

[0054] Table 1 siRNA sequences

[0055] 9) CCK8 detection Next, 5637 and T24 cells in the exponential growth phase were seeded at a density of 3000 cells / well in 96-well plates and cultured overnight. After treating the cells according to different grouping requirements, 10 μL of CCK8 solution was added to each well, and the plates were cultured for another 3 hours. Then, the culture plates were placed in a microplate reader (VLBLATGD2; Thermo Fisher Scientific), and the optical density was measured at 450 nm. The obtained data were used for statistical analysis.

[0056] 10) Flow cytometry detection of reactive oxygen species T24 and 5637 cells were collected during the exponential growth phase. After treatment according to grouping, cells were washed 2-3 times with pre-chilled phosphate-buffered saline (PBS). Following the instructions of the ROS detection kit (S0033S; Beyotime Biotech Inc., China), 2',7'-dichlorofluorescin diacetate probe was added to the cell suspension at a final concentration of 4 μmol / L. Cells were then incubated at 37°C and 5% CO2 for 30 minutes. After incubation, the fluorescence intensity of intracellular ROS was detected using flow cytometry (A24858; Thermo Fisher Scientific). The mean fluorescence intensity of ROS in each group was analyzed and calculated using ImageJ 1.5.2a software.

[0057] 11) Flow cytometry detection of apoptosis After treating cells in the exponential growth phase according to cell grouping, 5637 and T24 cells were collected after trypsin digestion, and the cell concentration was adjusted to approximately 1×10⁻⁶. 6 Cells / mL. Then, 100 μL of cell suspension was added to a flow cytometer tube. Following the operating procedure of the Annexin V-FITC / PI apoptosis detection kit (C1062L; Beyotime Biotech Inc.), 5 μL of Annexin V-FITC and 5 μL of PI dye were added sequentially, and the cells were incubated in the dark for 15 min. Changes in cell apoptosis were detected by flow cytometry. After data saving, statistical analysis was performed using FlowJo v10.8.1 software.

[0058] 12) Observe the structure of mitochondria using an electron microscope. 5637 and T24 cells in the exponential growth phase were treated according to their grouping. Cells were fixed with 2.5% glutaraldehyde for 4 hours, washed three times with PBS, and then dehydrated with a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%; 10 minutes each). After acetone replacement, the cells were embedded in epoxy resin. After the embedding blocks polymerized and solidified, ultrathin sections with a thickness of approximately 70 nm were cut using an ultramicrotome. The sections were placed on a copper grid and double-stained with uranium acetate and lead citrate for 15 minutes each time. Finally, the sections were observed and photographed using a transmission electron microscope (Talos F200X G2; Thermo Fisher Scientific).

[0059] 13) ATP content determination After grouping and treatment, 5637 and T24 cells in the exponential growth phase were centrifuged, and the supernatant was used for ATP content determination. Following the instructions of the ATP quantitative PCR kit (CB11233-Hu; COIBO BIO, China), the supernatant was mixed with the ATP detection reagent in a specific reaction system and incubated for 1 hour. Then, the optical density was measured at 450 nm using a microplate reader for subsequent analysis.

[0060] 14) Mitochondrial membrane potential detection After grouping and treating 5637 and T24 cells in the exponential growth phase, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanineiodide staining working solution was added according to the kit instructions (C2003S; Beyotime Biotech Inc.), and the cells were incubated at 37°C and 5% CO2 for 30 minutes. The cells were then washed 2-3 times with PBS and observed and photographed using a fluorescence microscope (AMF5000; Thermo Fisher Scientific). Changes in fluorescence intensity were analyzed using ImageJ 1.5.2a software.

[0061] 15) RT-qPCR Total RNA was extracted from 5637 and T24 cells in the exponential growth phase using TRIzol reagent (R1100; Beijing Solarbio Science & Technology Co., Ltd., China) according to the kit instructions. RNA concentration and purity were determined using a spectrophotometer (840-317500; Thermo Fisher Scientific). Reverse transcription was performed using a reverse transcription kit (RR037B; TaKaRa Biotechnology Co., Ltd., China). Specific primers were designed based on the mRNA sequences of the target and internal reference genes (Tables 2 and 3). For RT-PCR using cDNA as a template, the reaction system was prepared according to the instructions of the SYBR Green Real-Time PCR Kit (RR066B; TaKaRa Biotechnology Co., Ltd.). The reaction conditions were as follows: 42 °C for 5 min, 95 °C for 10 s, followed by 40 cycles, each consisting of 95 °C for 5 s and 60 °C for 30 s. After the reaction, 2... - The ΔΔCt method was used to analyze the relative expression levels of the target gene. Normalization was performed using an internal reference gene to compare the differences in target gene mRNA expression levels between different treatment groups and the control group.

[0062] Table 2 Primer sequences

[0063] Table 3 Primer Sequences

[0064] 16) Western blotting Next, 5637 and T24 cells in the exponential growth phase were transferred to centrifuge tubes according to their groups. Radioimmunoprecipitation assay buffer (P0013B; Beijing Solarbio Science & Technology Co., Ltd.) was added, and the cells were incubated on ice for 30 minutes. Protein was extracted from the supernatant. Protein concentration was determined using a BCA protein quantification kit (P0398L; Beijing Solarbio Science & Technology Co., Ltd.). Then, 30 μg of protein was added to each well of the electrophoresis tank. Electrophoresis was performed at 120 V for 90 minutes to separate the proteins under an electric field. The protein was then transferred to a polyvinylidene fluoride membrane. The transfer current was set to 260 mA for 1 hour. The membrane was incubated at 25 °C with 5% skim milk for 2 hours, and then incubated overnight at 4 °C with a suitable antibody. The antibody was diluted according to the manufacturer's instructions. The membrane was then incubated at 25 °C with the corresponding secondary antibody for 2 hours. After adding the ECL chemiluminescent substrate (P0018AS; Thermo Fisher Scientific), color development was performed using a chemiluminescence imager (A44241CFR; Thermo Fisher Scientific), following the manufacturer's instructions. The grayscale values ​​of the target protein bands were recorded and analyzed using ImageJ 1.52a software. The relative expression level of the target protein was normalized using the grayscale value of the internal control protein glyceraldehyde-3-phosphate dehydrogenase.

[0065] 17) Determination of extracellular acidification rate and oxygen consumption rate Cellular metabolic activity was detected using the Agilent Seahorse XFe96 analyzer system. Plasmid transfection, siRNA knockdown, or JuA treatment were performed according to the experimental design. On the day of assay, the culture medium in the cell culture plates was replaced with pre-prepared assay solutions using XF DMEM basal medium (103575-100, Agilent, China). The culture plates, along with sensor cartridges pre-hydrated and containing the Mitochondrial Stress Assay Kit (103015-100, Agilent, China) and the Glycolysis Rate Assay Kit (103344-100, Agilent, China), were then placed into the Seahorse XFe96 analyzer. The instrument operated according to the preset program. Real-time synchronous monitoring of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in each sample well using solid-state optical sensors enabled dynamic analysis of mitochondrial respiration and glycolysis functions.

[0066] 18) Statistical Analysis Data analysis was performed using SPSS software (version 20.0). All data are presented as mean ± standard error (SEM). Each mean was derived from at least three independent experiments. One-way ANOVA was used for comparisons among multiple groups, with Tukey post-hoc tests performed; t-tests were used for comparisons between two groups. Statistical significance was set at P < 0.05.

[0067] 2. Experimental Results 1) Screening for potential JuA targets among differentially expressed genes detected in bladder cancer. The GSE133624 dataset was downloaded from the Gene Expression Omnibus (GEO) database. Figure 1 A shows the principal component analysis results of the GSE133624 dataset, where red circles represent the case group (cancer tissue) and cyan circles represent the control group (Ctrls; normal tissue). Although most regions do not overlap, we observed partial overlap between the case and control groups, confirming specific differences in the transcriptomes of cancer and normal tissues. The volcano plot shows differentially expressed genes (DEGs). A total of 1845 DEGs were identified, of which 451 were upregulated and 1394 were downregulated. Figure 1 B). The heatmap shows the top 10 DEGs with the most significant upward and downward adjustments ( Figure 1 C). Pathway enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the phosphatidylinositol 3-kinase / protein kinase B signaling pathway, the cytokine-cytokine receptor pathway, and the cell adhesion molecule pathway. Figure 1 (D and E).

[0068] To investigate potential JuA drug targets among differentially expressed genes detected in bladder cancer, we searched for JuA targets in the Encyclopedia of Traditional Chinese Medicine database and analyzed their overlap with the detected differentially expressed genes. The results identified three potential targets (IL6, ATP1A2, and HSD11B1). Figure 1 F). Using the GENEMANIA database, we constructed a protein-protein interaction (PPI) network for these overlapping genes. Notably, these three potential target genes are associated with many other genes, such as IL6R, ATP1B2, and ATP1B1 (F). Figure 1 G).

[0069] To assess the clinical prognostic value of potential target genes (IL-6, ATP1A2, and HSD11B1), we constructed Kaplan-Meier survival curves based on the Cancer Genome Atlas (TCGA) database for overall survival (OS) analysis. Results showed no significant correlation between IL-6 levels and OS. However, high expression of ATP1A2 and HSD11B1 significantly reduced OS. Figure 1 The presence of H1A2 and HSD11B1 suggests that they may be potential biomarkers for poor prognosis. Currently, no studies have reported their specific roles in bladder cancer. Therefore, we focused on ATP1A2 in subsequent experiments.

[0070] 2) ATP1A2 overexpression promotes mitochondrial energy metabolism and inhibits apoptosis in 5637 cells. We assessed the expression level of ATP1A2 using different bladder cancer cell lines. Compared with RWPE-1 normal human prostate epithelial cells, the expression level of ATP1A2 was significantly upregulated in the UMUC3, 5637, RT4, and J83 bladder cancer cell lines, with the most significant upregulation observed in T24 cells. Figure 2 (A and 2B). To determine the effect of differential ATP1A2 expression on bladder cancer cells, we constructed an ATP1A2 overexpressing cell line in 5637 cells. RT-qPCR results showed that the ATP1A2 mRNA level was significantly increased in the oeATP1A2 group (transfected with the ATP1A2 overexpression plasmid), confirming the successful construction of a high ATP1A2 expression cell line. Figure 2 C). CCK8 assay results showed that ATP1A2 overexpression significantly improved cell viability (C). Figure 2 D) and reduced the apoptosis rate ( Figure 2 E). Western blotting analysis showed that ATP1A2 overexpression significantly reduced the expression level of Bcl-2-related X protein (Bax) and the cleaved-caspase-3 / caspase-3 ratio, and significantly increased the expression level of Bcl-2. Figure 2 The results (F) suggested that ATP1A2 overexpression inhibited apoptosis in 5637 cells. Functional experiments were then performed to investigate the relationship between ATP1A2 and mitochondrial energy metabolism. Detection using the 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanine iodide fluorescent probe showed that ATP1A2 overexpression significantly increased the mitochondrial membrane potential of 5637 cells (F). Figure 3 A) and intracellular ATP levels ( Figure 3 B), and significantly reduced reactive oxygen species (ROS) levels ( Figure 3C). Transmission electron microscopy (TEM) observation of cell ultrastructure revealed that under ATP1A2 overexpression conditions, mitochondria maintained intact morphology with orderly cristae arrangement, consistent with the morphological characteristics of the control group. Figure 3 D). We also observed changes in intracellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The results showed that, compared with the vector group, ATP1A2 overexpression significantly increased OCR and ECAR levels. These results suggest that overall cellular energy metabolism is enhanced. Figure 3 E). To further confirm the relationship with mitochondrial energy metabolism, we used Western blotting to detect the expression levels of proteins related to mitochondrial energy metabolism. The results showed that ATP1A2 overexpression significantly increased the relative protein levels of ATP1A2, ATP5A, COX1, COX2, NADH: ubiquinone oxidoreductase subunit A1 (UQCRC2), MTCO1, SDHB, and NDUFB8 (NDUFA1). Figure 3 (F and 3G). These results confirm that ATP1A2 overexpression enhances mitochondrial energy metabolism in 5637 cells.

[0071] 3) ATP1A2 knockdown inhibits mitochondrial energy metabolism in T24 cells and induces apoptosis. To verify the effect of ATP1A2 expression levels on bladder cancer cells, we constructed an ATP1A2 knockdown cell line of T24 cells. RT-qPCR results showed that siATP1A2-1, siATP1A2-2, and siATP1A2-3 all significantly reduced the relative expression level of ATP1A2 mRNA, with siATP1A2-1 and siATP1A2-2 showing the most significant reduction. Therefore, we selected siATP1A2-1 and siATP1A2-2 as interfering sequences for subsequent ATP1A2 knockdown cell model experiments. Figure 4 A). Knockdown of ATP1A2 significantly reduced cell viability ( Figure 4 B), and increased the apoptosis rate (B). Figure 4 C and D). Western blotting analysis showed that knockdown of ATP1A2 in T24 cells significantly increased Bax protein levels and the cleaved-caspase-3 / caspase-3 ratio, while significantly decreasing Bcl-2 protein levels. Figure 4 E), indicating that ATP1A2 knockdown induces apoptosis in T24 cells. ATP1A2 knockdown in T24 cells significantly reduces mitochondrial membrane potential (E). Figure 5 A) and intracellular ATP levels ( Figure 5 B), and increased ROS levels ( Figure 5C). Transmission electron microscopy further revealed that ATP1A2 knockdown led to characteristic morphological changes in T24 cell mitochondria, including damage to the mitochondrial membrane structure and disordered cristae arrangement. Figure 5 D). We also observed changes in intracellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) levels. The results showed that, compared to the siNC group, ATP1A2 knockdown significantly reduced OCR and ECAR levels. These results indicate that overall cellular energy metabolism is suppressed. Figure 5 E). Mitochondrial energy metabolism-related proteins were detected by Western blotting. ATP1A2 knockdown significantly reduced the relative expression levels of ATP1A2, ATP5A, COX1, COX2, NDUFA1, UQCRC2, MTCO1, SDHB, and NDUFB8. Figure 5 (F and 5G). These results suggest that ATP1A2 knockdown inhibits mitochondrial energy metabolism in T24 cells.

[0072] 4) JuA inhibits ATP1A2-induced apoptosis in T24 cells. We successfully demonstrated that ATP1A2 knockdown induces apoptosis and inhibits mitochondrial energy metabolism. The two-dimensional and three-dimensional molecular structures of JuA are shown below. Figure 6 As shown in Figure A. Molecular docking results indicate that the compound forms a stable interaction network with the target protein, forming six hydrogen bonds with key residues including D212, N382, K432, T445, and G447. These hydrogen bonds may involve both main chain and side chain atoms, indicating that the compound effectively occupies the protein's binding pocket and interacts specifically with key site residues. Furthermore, the docking score for this binding is 9.1 kcal / mol, reflecting a high theoretical binding affinity between the compound and the protein (…). Figure 6 B). To verify the potential targeting relationship between JuA and ATP1A2, we treated T24 cells with different concentrations of JuA (6.125, 12.5, 25, 50, and 100 μmol / L). The results showed that the viability of T24 cells decreased in a concentration-dependent manner with increasing JuA concentration. Different concentrations of JuA (25, 50, and 100 μmol / L) also gradually reduced the expression level of ATP1A2. Figure 6(C and D). To verify whether JuA exerts its anti-cancer effect by targeting and inhibiting ATP1A2, we transfected T24 cells with an ATP1A2 overexpression plasmid after JuA treatment. CCK8 assays and flow cytometry results showed that JuA significantly reduced T24 cell viability and induced apoptosis, while transfection with the ATP1A2 overexpression plasmid reversed these effects. Furthermore, Western blot analysis showed that JuA significantly upregulated Bax protein levels and the cleaved-caspase-3 / caspase-3 ratio in T24 cells, and significantly downregulated Bcl-2 protein levels (…). Figure 6 These results confirm that JuA targets and inhibits ATP1A2, thereby inducing apoptosis in T24 cells.

[0073] 5) JuA inhibits ATP1A2 expression and mitochondrial energy metabolism in T24 cells. Mitochondrial energy metabolism analysis showed that JuA significantly reduced mitochondrial membrane potential and intracellular ATP content in T24 cells, but increased reactive oxygen species (ROS) levels. However, ATP1A2 overexpression reversed these pharmacological effects of JuA. Figure 7 AC). Transmission electron microscopy (TEM) observations showed that JuA treatment induced characteristic morphological changes in T24 cell mitochondria, including damage to the mitochondrial membrane structure and disordered cristae arrangement. Figure 7 D). The results of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) assays showed that JuA significantly inhibited OCR and ECAR, thereby suppressing cellular energy metabolism. However, the inhibitory effect of JuA was reversed after overexpression of ATP1A2. Figure 7 E). Furthermore, ATP1A2 overexpression restored mitochondrial morphology. Subsequently, we performed Western blot experiments to detect the expression levels of proteins related to mitochondrial energy metabolism. JuA significantly reduced the relative expression levels of ATP1A2, ATP5A, COX1, COX2, NDUFA1, UQCRC2, MTCO1, SDHB, and NDUFB8 (E). Figure 7 (F and 7G). However, overexpression of ATP1A2 reversed this effect. These results confirm that JuA inhibits ATP1A2 expression, thereby suppressing mitochondrial energy metabolism in T24 cells.

[0074] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of jujube seed saponin A in the preparation of drugs for treating bladder cancer.

2. The application according to claim 1, characterized in that, The medication also includes other drugs for treating bladder cancer.

3. The application according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include excipients, buffers, surfactants, and preservatives.

4. The application according to claim 3, characterized in that, The dosage forms of the drug include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

5. The application according to claim 4, characterized in that, The gastrointestinal dosage forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets. Preferably, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

6. A drug for treating bladder cancer, characterized in that, The drug includes jujube seed saponin A.

7. A method for inhibiting bladder cancer cell viability / promoting bladder cancer cell apoptosis in vitro, characterized in that, The method includes applying jujube seed saponin A; Preferably, the bladder cancer cells are T24 cells.

8. A method for preparing cells with reduced ATP1A2 levels, characterized in that, The method includes applying jujube seed saponin A; Preferably, the cells are bladder cancer cells; Preferably, the bladder cancer cells are T24 cells.

9. Application of jujube seed saponin A in the preparation of products that regulate the levels of Bcl-2 and mitochondrial energy metabolism-related proteins in cells; Preferably, the mitochondrial energy metabolism-related proteins include any one or more of ATP1A2, ATP5A, COX1, COX2, NDUFA1, UQCRC2, MTCO1, SDHB, and NDUFB8; Preferably, the cells are bladder cancer cells; Preferably, the bladder cancer cells are T24 cells.

10. Application of jujube seed saponin A in the preparation of products that inhibit mitochondrial energy metabolism; Preferably, the inhibition of mitochondrial energy metabolism includes reducing any one or more of the following: mitochondrial membrane potential, ATP content, oxygen consumption rate, and extracellular acidification rate. Preferably, the inhibition of mitochondrial energy metabolism is the inhibition of mitochondrial energy metabolism in bladder cancer cells; Preferably, the bladder cancer cells are T24 cells.

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