Application of combination of dioscin and chidamide in diffuse large B-cell lymphoma

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of dioscin combined with chidamide in diffuse large B-cell lymphoma. Specifically, research finds that the dioscin can significantly inhibit DLBCL cell proliferation and induce cell cycle arrest and apoptosis by up-regulating PINK1 / Parkin mediated mitochondrial autophagy and enhancing H3K27 acetylation mediated epigenetic regulation. When the chidamide and the dioscin are combined for use, a synergistic effect can be generated, and by further increasing the H3K27ac level, the cell apoptosis induction effect is enhanced, and the in-vivo and in-vitro anti-tumor activity on DLBCL is remarkably improved. The invention provides a novel and efficient DLBCL combined treatment scheme, and provides a new pharmaceutical composition and theoretical support for clinical diagnosis and treatment of DLBCL, so that the application has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of diosgenin combined with chidamide in diffuse large B-cell lymphoma. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma in adults worldwide, accounting for approximately one-third of all non-Hodgkin's lymphoma diagnoses annually, and posing a significant socioeconomic burden affecting millions of people. The CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen has been used for over 40 years. Rituximab was approved by the U.S. Food and Drug Administration in 2006 for use in combination with CHOP as first-line treatment for DLBCL patients. Since then, the R-CHOP regimen has become the standard treatment for newly diagnosed DLBCL patients; however, approximately 30%–40% of patients remain unresponsive to first-line therapy or eventually relapse into refractory disease, with a 5-year survival rate of less than 60%. Therefore, exploring new drugs and combination therapy strategies is of great significance for improving the clinical benefits for DLBCL patients.

[0004] Chidamide is a novel oral selective histone deacetylase (HDAC) inhibitor, a benzamide class, that inhibits class I HDAC1, HDAC2, and HDAC3, as well as class IIb HDAC10. It was approved by the China National Medical Products Administration in 2014 for the treatment of relapsed / refractory peripheral T-cell lymphoma. Chidamide can induce tumor cell apoptosis, inhibit proliferation, and affect the immune microenvironment and multiple signaling pathways by regulating epigenetic modifications. In recent years, it has also shown potential efficacy in preclinical studies of various lymphomas, including DLBCL. However, its antitumor activity as a monotherapy is relatively low, necessitating further exploration of novel chidamide-based combination therapies to improve clinical efficacy in lymphoma patients.

[0005] Dioscin is a natural steroidal saponin widely found in plants of the Dioscorea genus and is also an active ingredient in some traditional Chinese medicines, including Liuwei Dihuang Decoction. Clinically, dioscin tablets are used to treat coronary artery insufficiency and to treat coronary heart disease and angina pectoris. It also has some efficacy in treating conditions accompanied by hypertension, high triglycerides, and high cholesterol. Pharmacological experiments have shown that dioscin has anti-tumor, anti-inflammatory, immunomodulatory, lipid-lowering, antiviral, antifungal, and anti-allergic effects. In addition, some recent studies have suggested that dioscin can regulate metabolic diseases including diabetes, osteoporosis, obesity, and hyperuricemia, and exert anti-tumor effects through the regulation of lipid metabolism, induction of apoptosis, and ferroptosis. Currently, there are no reports on the use of dioscin in DLBCL, either domestically or internationally. Summary of the Invention

[0006] To address the aforementioned limitations of existing technologies, this invention provides the application of diosgenin combined with chidamide in diffuse large B-cell lymphoma (DLBCL). Specifically, this invention has found that diosgenin can significantly inhibit the growth of DLBCL in a time- and concentration-dependent manner. In vitro experiments show that diosgenin can promote mitophagy in DLBCL cells and exert anti-tumor effects by upregulating H3K27 acetylation, thereby producing a synergistic anti-tumor effect. Moreover, the combination of diosgenin and chidamide exhibits good anti-tumor effects in both DLBCL cells and mouse models. Based on the above research findings, this invention is thus completed.

[0007] Specifically, the present invention relates to the following technical solutions: In a first aspect, the invention provides the use of diosgenin in combination with chidamide in the preparation of a therapeutic agent for diffuse large B-cell lymphoma.

[0008] The molar ratio of diosgenin to chidamide is 1-3:1-3.

[0009] Specifically, this invention demonstrates through research that diosgenin can significantly inhibit the growth of DLBCL in a time- and concentration-dependent manner. In vitro experiments show that diosgenin can promote mitophagy in DLBCL cells and exert its anti-tumor effect by upregulating H3K27 acetylation. Moreover, diosgenin combined with chidamide exhibits good anti-tumor effects in both DLBCL cells and mouse models.

[0010] Therefore, the above-mentioned diosgenin combined with chidamide can be used to treat diffuse large B-cell lymphoma. Meanwhile, according to the present invention, the concept of "treatment" means any measure applicable to the treatment of tumors and related diseases, or preventive treatment of such diseases or their symptoms, or prevention of recurrence of such diseases, such as recurrence after the end of a treatment period or treatment of symptoms of an already occurring disease, or preemptive intervention to prevent, suppress, or reduce the occurrence of such diseases or symptoms.

[0011] A second aspect of the invention provides the use of diosgenin or diosgenin in combination with chidamide in any one or more of the following: (b1) Prepare a product that inhibits the proliferation of diffuse large B-cell lymphoma cells; (b2) Prepare products that induce cell cycle arrest in diffuse large B-cell lymphoma; (b3) Prepare products that induce apoptosis in diffuse large B-cell lymphoma cells; (b4) Prepare products that induce oxidative stress in diffuse large B-cell lymphoma cells and cause mitochondrial damage; (b5) Prepare products that reduce mitochondrial membrane potential in diffuse large B-cell lymphoma cells and promote mitochondrial autophagy; (b6) Prepare products that regulate the histone acetylation levels in diffuse large B-cell lymphoma cells; (b7) To prepare a product that increases the sensitivity of diffuse large B-cell lymphoma to chidamide; (b8) Prepare products for treating diffuse large B-cell lymphoma.

[0012] In (b1)-(b8), the product may be a drug or a general test reagent for non-medical purposes. The test reagent may be used for basic research to construct relevant tumor cells or animal models, and then to study related mechanisms such as the tumorigenesis and development mediated by mitochondrial autophagy in tumor cells.

[0013] In (b2), the diffuse large B-cell lymphoma cell cycle is arrested in the G0 / G1 phase.

[0014] In (b4), the induction of oxidative stress in diffuse large B-cell lymphoma cells is specifically manifested as a significant increase in intracellular SOX levels; Induced mitochondrial damage in diffuse large B-cell lymphoma cells is specifically manifested as a decrease in intracellular TOM20 expression.

[0015] In (b5), promoting mitochondrial autophagy in diffuse large B-cell lymphoma cells is specifically manifested by upregulating the expression levels of intracellular PINK1, Parkin, and LC3B, and downregulating the expression levels of p62, HSP60, and TOM20.

[0016] In (b6), the regulation of histone acetylation levels in diffuse large B-cell lymphoma cells is specifically manifested as increasing the level of H3K27ac in diffuse large B-cell lymphoma cells.

[0017] When the product is a pharmaceutical product, the pharmaceutical product further includes at least one non-pharmaceutical active ingredient. The non-pharmaceutical active ingredient includes pharmaceutically acceptable carriers, excipients, and / or diluents.

[0018] A third aspect of the present invention provides a pharmaceutical composition in which the active ingredient comprises diosgenin in combination with chidamide; wherein the molar ratio of diosgenin to chidamide is 1-3:1-3.

[0019] The pharmaceutical composition has a synergistic effect against diffuse large B-cell lymphoma.

[0020] The pharmaceutical composition may also contain at least one non-pharmaceutical active ingredient. The non-pharmaceutical active ingredient includes pharmaceutically acceptable carriers, excipients, and / or diluents.

[0021] In another specific embodiment of the present invention, the non-pharmaceutical active ingredient includes: Pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids; pharmacologically harmless salts, flavorings, vitamins, antioxidants, and stabilizers and / or preservatives.

[0022] The dosage forms of the pharmaceutical preparations include: liquid dosage forms, solid dosage forms, external preparations, and sprays; In another specific embodiment of the present invention, the following dosage forms are included: true solutions, colloids, microparticles, emulsions, suspensions, tablets, capsules, pellets, aerosols, pills, powders, solutions, granules, suppositories, lyophilized powder injections, inclusion complexes, fillers, patches, and liniments.

[0023] A fourth aspect of the present invention provides a method for treating diffuse large B-cell lymphoma, the method comprising administering the above-described pharmaceutical composition to a subject.

[0024] The subject refers to an animal that is already being treated, observed, or experimented on, preferably a mammal, and most preferably a human.

[0025] The beneficial technical effects of one or more of the above technical solutions: The above-mentioned technical solution is the first report demonstrating that diosgenin can significantly inhibit the growth of DLBCL in a time- and concentration-dependent manner. Simultaneously, diosgenin can promote mitophagy in DLBCL cells and exert anti-tumor effects by upregulating H3K27 acetylation. Furthermore, diosgenin combined with chidamide exhibited good anti-tumor effects in both DLBCL cells and mouse models.

[0026] The above-mentioned technical solutions can promote in-depth research and clinical application of the combined drug regimen of diosgenin and chidamide, and provide a solid theoretical basis and experimental foundation for constructing new and efficient combined drug strategies and optimizing clinical diagnosis and treatment of DLBCL. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 The diagram shows the molecular structure and molecular formula of diosgenin (A) and chidamide (B) in the embodiments of the present invention.

[0029] Figure 2 This embodiment of the invention demonstrates the level of DLBCL cell proliferation inhibition by diosgenin.

[0030] Figure 3 In this embodiment of the invention, diosgenin induces cell cycle arrest in DLBCL cells.

[0031] Figure 4 This invention provides an example of diosgenin-induced apoptosis in DLBCL cells.

[0032] Figure 5 In this embodiment of the invention, diosgenin treatment resulted in increased oxidative stress (A) and decreased mitochondrial numbers in DLBCL cells (B).

[0033] Figure 6 In this embodiment of the invention, diosgenin reduces mitochondrial membrane potential in DLBCL cells (A) and promotes mitochondrial autophagy (B).

[0034] Figure 7 This invention illustrates the molecular mechanism by which diosgenin promotes mitochondrial autophagy in DLBCL cells. A represents the Western blotting results of diosgenin-mediated regulation of mitochondrial autophagy-related molecules in DLBCL cells, and B shows that the mitochondrial autophagy inhibitor Mdivi-1 can partially restore the inhibitory effect of diosgenin on DLBCL cell proliferation.

[0035] Figure 8This embodiment of the invention illustrates the regulatory effect of diosgenin on histone acetylation levels in DLBCL cells; wherein, A represents the significant increase in H3K27ac levels in DLBCL cells by diosgenin, and B represents the partial reversal of the inhibitory effect of diosgenin on DLBCL cell proliferation by histone acetylation inhibitor C646.

[0036] Figure 9 This invention relates to the effects of diosgenin on increasing the sensitivity of chidamide and the combined use of these drugs on the H3K27ac level in DLBCL cells. Specifically, A represents the index (CI) analysis of different concentrations of diosgenin and chidamide; B represents the effect of different drug administrations on the expression level of histone H3K27ac.

[0037] Figure 10 In this embodiment of the invention, diosgenin and chidamide are used in combination to promote apoptosis in DLBCL cells.

[0038] Figure 11 This invention illustrates the combined anti-lymphoma effect of diosgenin and chidamide in a DLBCL mouse xenograft model. A represents tumor growth curves in different groups of mice; B represents tumor images after dissection in different groups of mice; and C represents tumor signal intensity after in vivo fluorescence imaging in different groups of mice. Detailed Implementation

[0039] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0040] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0041] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0042] Example Experimental methods 1. Cell Culture The human DLBCL cell lines OCI-LY1 and OCI-LY10 used in this study were purchased from ATCC. Cells were cultured in IMDM complete medium containing 10% fetal bovine serum at 37°C in a cell culture incubator containing 5% CO2. The medium was changed and passaged every 2-3 days, and cells with good growth were used for subsequent experiments.

[0043] 2. Cell counting kit-8 (CCK-8) was used to detect the proliferation level of DLBCL cells. Diosgenin, chidamide, Mdivi-1, and C646 powder were dissolved in DMSO to prepare a 10 mM stock solution. DLBCL cells in the logarithmic growth phase and in good condition were collected, centrifuged, and the supernatant was removed. The cells were resuspended in IMDM complete medium and counted under a microscope, at a density of 10 cells per well. 4 DLBCL cells were seeded into 96-well cell culture plates at a concentration ratio of 1 cell / 90 μL. 10 μL of different drug concentrations were added according to the concentration conversion formula, with blank wells and three duplicate wells. The plates were gently shaken to mix, and the 96-well plates were placed in a 37°C cell culture incubator containing 5% CO2 for further culture. After 48 hours, 10 μL of CCK8 reagent was added to each well, taking care not to generate air bubbles. The plates were incubated at 37°C in the dark for 1-4 hours. The absorbance (OD) value at 450 nm was then measured using a microplate reader, and the growth curve of DLBCL cells was plotted based on the results.

[0044] 3. CellTiter-Glo Luminescent assays (CTG) were used to detect the viability level of DLBCL cells. Diosgenin and chidamide powders were dissolved in DMSO to prepare a 10 mM stock solution. DLBCL cells in the logarithmic growth phase and in good condition were collected, centrifuged, and the supernatant was removed. The cells were resuspended in IMDM complete medium and counted under a microscope, at a concentration of 10 cells per well. 4 DLBCL cells were seeded into 384-well cell culture plates at a ratio of 1 cell / 25 μL. The drug was diluted to 125 μM working solution with IMDM, and 1 μL of the drug was added to each well. Blank wells and three duplicate wells were set up. After gently shaking to mix, the 384-well plates were placed in a cell culture incubator at 37°C with 5% CO2 for further culture. After 48 hours, 25 μL of CTG reagent was added to each well, taking care not to generate air bubbles. The plates were shaken at room temperature in the dark for 10 min. The luminescence value (RLU) of each well was then recorded using a microplate luminescence detector, and the viability of DLBCL cells was calculated based on the detection results.

[0045] 4. Cell cycle analysis (flow cytometry analysis of cell cycle) Collect processed DLBCL cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells in pre-chilled sterile PBS, centrifuge again at 1000 rpm for 5 min, discard the supernatant, and repeat the washing twice. Then resuspend the cells in 500 μL of pre-chilled PBS, and add the cell suspension dropwise to an EP tube containing 1200 μL of pre-chilled anhydrous ethanol, gently pipetting to mix and ensure complete cell fixation. Store the samples at -20°C protected from light for at least 24 hours. After fixation, centrifuge the cells at 3000 rpm for 5 min, discard the supernatant, resuspend the cells in pre-chilled PBS, and wash twice. Then perform cell counting and adjust the cell volume to 1 × 10⁻⁶ cells / mL. 6 For each sample group, resuspend the cells in 500 μL of PI / RNase staining buffer. After incubating the samples at room temperature in the dark for 15-30 min, filter the cell suspension through a 400-mesh filter and transfer it to labeled flow cytometry tubes for cell cycle distribution analysis.

[0046] 5. Apoptosis detection (Flow cytometry analysis of apoptosis) Collect processed DLBCL cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells in pre-chilled sterile PBS, centrifuge again at 1000 rpm for 5 min, discard the supernatant, and repeat the washing process twice. Then resuspend the cells in 500 μL of binding buffer, count them under a microscope, and adjust the cell concentration to 1 × 10⁻⁶. 6 Cells / mL. The cell suspension was filtered through a 400-mesh filter and transferred to labeled flow cytometry tubes. Under dark conditions, 5 μL each of Annexin V-PE and 7-AAD staining solution were added to each tube, and the mixture was gently pipetted to mix. After incubation at room temperature in the dark for 15-30 min, flow cytometry was immediately performed to analyze the apoptosis level.

[0047] 6. Construction of a DLBCL xenograft mouse model and in vivo imaging of the mice. The 3-4 week old male SCID Beige nude mice used in this study were purchased from Beijing Vital River Laboratory Animal Co., Ltd. They were housed in a sterile environment with sterile drinking water, feed, and bedding, maintaining a sterile, constant temperature (25℃), and constant humidity (70%). The animal research process strictly followed the ARRIVE guidelines and the principles of the Animal Protection and Research Ethics Committee of Shandong Provincial Hospital. The experimental mice were randomly divided into four groups: a control group, a diosgenin group, a chidamide group, and a diosgenin combined with chidamide group, with four mice in each group. DLBCL cells in the logarithmic growth phase and in good condition were collected and the cell density was adjusted to 1×10⁻⁶. 8Diosgenin was added to an equal volume of Matrigel and mixed thoroughly. 200 μL of the mixture was then inoculated subcutaneously into the right lateral hind limb of mice. An oval wheal was visibly formed. The mice were then kept in a sterile environment. Once the tumor protruded above the skin surface, drug treatment was initiated, administering diosgenin (80 mg / kg) and chidamide (10 mg / kg) once daily via gavage. Tumor size was measured and recorded daily using calipers. a = long diameter of the tumor, b = short diameter of the tumor, tumor volume (V) = (a × b) / (a × b) / (b ... 2 A tumor growth curve was plotted using a factor of 0.5. After 21 days, in vivo imaging was performed on mice to assess the drug's effectiveness. Mice were fasted for 30 minutes to 1 hour before imaging to reduce gastrointestinal interference. Mice were anesthetized with 2-3% isoflurane inhalation and fixed to the imaging platform, ensuring the tumor area faced the detector. Fluorescence emission mode was selected, and the excitation source and emission filter were adjusted to acquire whole-body scan images. The imaging system software was used to quantitatively analyze the tumor area signal, obtaining total luminous flux or average radiation intensity data. Signal intensities from different groups of mice were statistically compared. After imaging, mice were euthanized by cervical dislocation, and intact subcutaneous tumors were dissected and photographed for preservation. Mouse tissues were collected and stored in 4% paraformaldehyde and -80°C ultra-low temperature freezers for subsequent experiments.

[0048] 7. Protein extraction and Western blotting analysis. Total protein was extracted by collecting treated DLBCL cells, resuspending and washing the cells with pre-cooled PBS, repeating the process three times. After centrifugation to remove the supernatant, an appropriate amount (40-150 μL) of protein lysis buffer (RIPA:PMSF:phosphatase inhibitor volume ratio of 100:1:1) was added according to the size of the cell clumps. The mixture was vortexed and incubated on ice for 30 min. After centrifugation at 12000 rpm for 30 min at 4 °C, the supernatant containing protein was collected. Nuclear proteins were extracted from treated DLBCL cells. Cells were resuspended and washed with pre-chilled PBS, repeated three times. After centrifugation to remove the supernatant, 200 μL of cytoplasmic protein extraction reagent A (containing PMSF) was added to every 20 μL of cell pellet. The mixture was incubated on ice for 10-15 min, followed by 10 μL of cytoplasmic protein extraction reagent B, incubated on ice for 1 min, and centrifuged at 12000g for 5 min at 4°C. All remaining supernatant was aspirated, and 50 μL of nuclear protein extraction reagent (containing PMSF) was added. The mixture was incubated on ice for 30 min, and centrifuged at 12000g for 10 min at 4°C. Protein concentration was determined using the BCA method. The supernatant was then mixed with loading buffer at a 3:1 volume ratio and incubated at 100°C for 10 min to denature the proteins. The mixture was then stored at -80°C for later use. Prepare a 7.5-12% electrophoresis gel according to the molecular weight of the target protein. Place it in an electrophoresis tank and add an appropriate amount of electrophoresis buffer. Add 40 μg of protein or 4 μL of protein marker to each well sequentially. Perform electrophoresis at a constant voltage of 200V. Determine the electrophoresis time based on the position of the protein marker and bromophenol blue. Cut a PVDF membrane to the size corresponding to the gel beforehand and soak it in methanol for 1-2 min. Immerse the cut gel, PVDF membrane, and transfer filter paper in transfer buffer, and place them from bottom to top as follows: transfer filter paper-PVDF membrane-gel-transfer filter paper. Carefully remove air bubbles and place it in a semi-dry transfer tank. Transfer the membrane at a constant voltage of 10V for 30 min. Place the PVDF membrane in 10% skim milk prepared with TBST and block it at room temperature for 1-2 hours. Then, place the PVDF membrane in a primary antibody solution of an appropriate concentration prepared with primary antibody dilution buffer and incubate it overnight on a shaker at 4°C. After washing the PVDF membrane three times with TBST, it was placed in a secondary antibody solution prepared with 10% skim milk and incubated at room temperature for 1 hour. The PVDF membrane was washed again, and a pre-prepared developing solution (A solution: B solution = 1:1) was added. The membrane was then imaged using an Amersham Imager 600 gel imaging instrument, and the gray values ​​of the bands were analyzed using ImageJ software to assess the expression level of the target molecule.

[0049] 8. Immunofluorescence Staining Collect processed DLBCL cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Wash the cells twice with pre-chilled sterile PBS, resuspend them in pre-chilled PBS, count them under a microscope, and adjust the cell concentration to 1×10⁶ cells / mL. 6 Cells / mL. Add 700 μL of cell suspension to the circular well of a plastic slide for a cell smear machine, centrifuge at 800 rpm for 5 min to ensure even cell spread. After observing the cell morphology and distribution under a microscope, add approximately 1 mL of sterile PBS solution to the slide and incubate at room temperature for 5 min. After aspirating excess liquid, add approximately 1 mL of 4% paraformaldehyde fixative and fix at room temperature for 15 min. After fixation, wash the slide in PBS for 5 min each time, for a total of 3 times. After aspirating the surface moisture, add 0.2% Triton X-100 for cell membrane permeabilization treatment and incubate at room temperature for 10 min, then wash 3 times with PBS. Finally, add 2% goat serum blocking solution to the slide and incubate at 37°C for 60 min. After removing the blocking solution, add diluted primary antibody solutions (TOM20 antibody, 1:200; LC3B antibody, 1:200), place the slide in a humidified chamber, and incubate overnight (at least 16 hours) at 4°C in the dark. The next day, wash three times with PBS for 5 minutes each time, aspirate excess liquid, add prepared secondary antibody solutions (1:200), and incubate at 37°C in the dark for 60 minutes. Wash three more times with PBS, aspirate excess liquid, add 1–2 drops of anti-fluorescence attenuation mounting medium containing DAPI, and slowly cover with a coverslip, avoiding air bubbles. Finally, observe the cells using a laser confocal microscope, randomly selecting five fields of view from each slide for photographing and data recording.

[0050] 9. Mitochondrial reactive oxygen species (ROS) detection (MitoSOX™ Red staining method) Collect processed DLBCL cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Wash the cells twice with pre-chilled sterile PBS, resuspend them in pre-chilled PBS, count them under a microscope, and adjust the cell concentration to 1×10⁶ cells / mL. 6Cells / mL. Add 700 μL of cell suspension to the circular aperture of a plastic slide for a cell smear machine, centrifuge at 800 rpm for 5 min to ensure even cell spread. After observing the cell morphology and distribution under a microscope, add approximately 1 mL of sterile PBS solution to the slide and incubate at room temperature for 5 min. After aspirating excess liquid, add approximately 1 mL of 4% paraformaldehyde fixative and fix at room temperature for 15 min. After fixation, wash the slide in PBS for 5 min each time, for a total of 3 times. After aspirating the surface moisture, add an appropriate amount of pre-prepared MitoSOX™ Red staining working solution and incubate at 37°C for 60 min. Wash three more times with PBS, aspirate excess liquid, add 1–2 drops of DAPI-containing anti-fluorescence attenuation mounting medium, and slowly cover with a coverslip, avoiding air bubbles. Finally, observe the cells using a laser confocal microscope, randomly selecting 5 fields of view from each slide for photographing and data recording.

[0051] 10. Mitochondrial membrane potential detection (JC-1 staining method) Collect processed DLBCL cells, taking approximately 1 × 10⁻⁶ cells. 5 Up to 6×10 5 Cells were resuspended in 0.5 mL of cell culture medium containing serum and phenol red. 0.5 mL of JC-1 staining working solution was added to the cell suspension, and the mixture was gently inverted to mix. The cells were then incubated at 37°C for 20 min in a 5% CO2 incubator. During incubation, an appropriate amount of JC-1 staining buffer (1×) was prepared by adding 4 mL of distilled water to every 1 mL of JC-1 staining buffer (5×) and kept on ice. After incubation, the cells were centrifuged at 600 g at 4°C for 4 min, and the supernatant was discarded. The cells were then washed twice with pre-chilled JC-1 staining buffer (1×): each time, 1 mL of JC-1 staining buffer (1×) was added to resuspend the cells, and the cells were centrifuged at 600 g at 4°C for 4 min, and the supernatant was discarded. After washing, the cells were resuspended with an appropriate amount of JC-1 staining buffer (1×) and observed under a fluorescence microscope. The ratio of JC-1 aggregated red fluorescence to monomeric green fluorescence was used to assess changes in mitochondrial membrane potential.

[0052] result Figure 1 The schematic diagrams and molecular formulas of diosgenin and chidamide are shown.

[0053] Figure 2 The study demonstrated the inhibitory effect of diosgenin on the proliferation of DLBCL cells. The results showed that diosgenin significantly inhibited the proliferation of DLBCL cells in a concentration- and time-dependent manner. These results indicate that diosgenin possesses a significant anti-lymphoma proliferative effect in vitro.

[0054] Figure 3The study demonstrated that diosgenin induces cell cycle arrest in DLBCL cells. The results showed that diosgenin can induce cell cycle arrest in the G0 / G1 phase of DLBCL cells, and the arrest effect is concentration-dependent.

[0055] Figure 4 The study demonstrated that diosgenin induced apoptosis in DLBCL cells. The results showed that diosgenin could induce apoptosis in DLBCL cells in a concentration-dependent manner. These results indicate the in vitro anti-lymphoma activity of diosgenin.

[0056] Figure 5 The study showed that diosgenin treatment increased oxidative stress and reduced the number of mitochondria in DLBCL cells. Figure 5 Results A showed that intracellular SOX levels were significantly increased after diosgenin treatment, indicating enhanced cellular oxidative stress. Figure 5 Results B showed that immunofluorescence assays revealed a significant decrease in TOM20 expression after diosgenin treatment, confirming a reduction in mitochondrial numbers. These results suggest that diosgenin can induce oxidative stress and lead to mitochondrial damage.

[0057] Figure 6 The study showed that diosgenin reduced mitochondrial membrane potential in DLBCL cells and promoted mitophagy. Figure 6 A shows the results of JC-1 staining. Dioscorea saponin treatment significantly reduced mitochondrial membrane potential, indicating that mitochondrial membrane integrity was impaired, further proving that dioscorea saponin can induce mitochondrial dysfunction. Figure 6 Results B showed a significant enhancement in the colocalization signal between the autophagy marker protein LC3B and the mitochondrial outer membrane protein TOM20, suggesting enhanced mitophagy. These results indicate that diosgenin can induce mitochondrial dysfunction and activate mitophagy.

[0058] Figure 7 This study revealed the molecular mechanism by which diosgenin promotes mitochondrial autophagy in DLBCL cells. Figure 7 A shows the results of Western blotting, which indicated that diosgenin significantly upregulated the expression levels of PINK1, Parkin, and LC3B. PINK1 is a sensory protein for mitochondrial damage, Parkin is a key E3 ligase mediating mitochondrial ubiquitination and initiating mitophagy, and LC3B is an important marker of autophagosome formation. Conversely, diosgenin downregulated the expression levels of p62, HSP60, and TOM20. The decrease in p62 suggests enhanced autophagic flux, HSP60 is a mitochondrial matrix chaperone protein, and the decrease in TOM20 is a marker protein of the mitochondrial outer membrane, indicating mitochondrial clearance by autophagy. Figure 7Results B showed that the mitophagy inhibitor Mdivi-1 could partially restore the inhibitory effect of diosgenin on DLBCL cell proliferation. These results suggest that diosgenin exerts its anti-DLBCL effect by promoting PINK1 / Parkin-mediated mitophagy.

[0059] Figure 8 This study demonstrated the regulatory effect of diosgenin on histone acetylation levels in DLBCL cells. Figure 8 The results showed that diosgenin significantly increased the level of H3K27ac in DLBCL cells; Figure 8 B showed that the histone acetylation inhibitor C646 could partially reverse the inhibitory effect of diosgenin on DLBCL cell proliferation. These results suggest that diosgenin exerts its anti-DLBCL effect by enhancing H3K27ac-mediated epigenetic regulation.

[0060] Figure 9 The study showed that diosgenin increases the sensitivity of chidamide and that the combination therapy has an effect on the H3K27ac level in DLBCL cells. Figure 9 In A, the CTG experiment results showed that the combination index (CI) analysis of different concentrations of diosgenin and chidamide showed that the two had a synergistic effect (CI<1). Figure 9 B showed that western blotting results indicated that the level of histone H3K27ac was increased when diosgenin was used in combination with chidamide compared with the drug alone and the control group.

[0061] Figure 10 Diosgenin combined with chidamide promotes apoptosis in DLBCL cells. Results showed that both diosgenin and chidamide alone promoted apoptosis in DLBCL cells, while combined administration significantly increased apoptosis levels, indicating a synergistic effect between the two in inducing apoptosis.

[0062] Figure 11 This study demonstrated the combined anti-lymphoma effect of diosgenin and chidamide in a DLBCL mouse xenograft model. Figure 11 A shows the tumor growth curves of different groups of mice. Both diosgenin and chidamide can inhibit the growth of lymphoma. The addition of diosgenin can enhance the in vivo antitumor effect of chidamide. Figure 11 B shows images of tumors after dissection in mice from different groups. The tumor size and volume in the combined drug group were significantly smaller than those in the control group and the single drug treatment group. Figure 11 C shows the tumor signal intensity after in vivo fluorescence imaging in different groups of mice. The results show that the tumor signal was significantly weakened in the combined drug group, further demonstrating the synergistic antitumor effect of diosgenin and chidamide in vivo. The results demonstrate the combined in vivo antitumor effect of diosgenin and chidamide.

[0063] In summary, diosgenin significantly inhibits DLBCL cell proliferation and induces cell cycle arrest and apoptosis by upregulating PINK1 / Parkin-mediated mitophagy and enhancing H3K27 acetylation-mediated epigenetic regulation. The combined use of chidamide and diosgenin produces a synergistic effect, further increasing H3K27ac levels and enhancing apoptosis induction, thus significantly improving the in vitro and in vivo antitumor activity against DLBCL. This invention provides a novel and highly effective combination therapy for DLBCL, offering new drug combinations and theoretical support for the clinical diagnosis and treatment of DLBCL.

[0064] Matters not covered in this invention are common knowledge.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of diosgenin combined with chidamide in the preparation of a therapeutic drug for diffuse large B-cell lymphoma.

2. The application as described in claim 1, characterized in that, The molar ratio of diosgenin to chidamide is 1-3:1-3.

3. The use of diosgenin or diosgenin combined with chidamide in any one or more of the following: (b1) Prepare a product that inhibits the proliferation of diffuse large B-cell lymphoma cells; (b2) Prepare products that induce cell cycle arrest in diffuse large B-cell lymphoma; (b3) Prepare products that induce apoptosis in diffuse large B-cell lymphoma cells; (b4) Prepare products that induce oxidative stress in diffuse large B-cell lymphoma cells and cause mitochondrial damage; (b5) Prepare products that reduce mitochondrial membrane potential in diffuse large B-cell lymphoma cells and promote mitochondrial autophagy; (b6) Prepare products that regulate the histone acetylation levels in diffuse large B-cell lymphoma cells; (b7) To prepare a product that increases the sensitivity of diffuse large B-cell lymphoma to chidamide; (b8) Prepare products for treating diffuse large B-cell lymphoma.

4. The application as described in claim 3, characterized in that, In (b1)-(b8), the product is a drug or a general testing reagent for non-medical purposes.

5. The application as described in claim 3, characterized in that, In (b2), the diffuse large B-cell lymphoma cell cycle is arrested in the G0 / G1 phase.

6. The application as described in claim 3, characterized in that, In (b4), the induction of oxidative stress in diffuse large B-cell lymphoma cells is specifically manifested as a significant increase in intracellular SOX levels; Induced mitochondrial damage in diffuse large B-cell lymphoma cells is specifically manifested as a decrease in intracellular TOM20 expression.

7. The application as described in claim 3, characterized in that, In (b5), promoting mitochondrial autophagy in diffuse large B-cell lymphoma cells is specifically manifested by upregulating the expression levels of intracellular PINK1, Parkin, and LC3B, and downregulating the expression levels of p62, HSP60, and TOM20.

8. The application as described in claim 3, characterized in that, In (b6), the regulation of histone acetylation levels in diffuse large B-cell lymphoma cells is specifically manifested as increasing the level of H3K27ac in diffuse large B-cell lymphoma cells.

9. A pharmaceutical composition, characterized in that, Its active ingredients include diosgenin combined with chidamide; wherein the molar ratio of diosgenin to chidamide is 1-3:1-3.

10. The use of the pharmaceutical composition of claim 9 in the preparation of a synergistic anti-diffuse large B-cell lymphoma drug.

Images