Application of pharmaceutical composition in treatment of diffuse large B-cell lymphoma

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a pharmaceutical composition in treatment of diffuse large B-cell lymphoma. Specifically, two groups of pharmaceutical compositions, namely the magnolin and the vonacrah as well as the masitinib and the gemcitabine hydrochloride, are found, and the molar ratio of the magnolin to the vonacrah is (40-120): (0.2-0.8). The molar ratio of masitinib to gemcitabine hydrochloride is (1-10): (1-10). Research finds that the pharmaceutical composition can synergistically inhibit growth of DLBCL cells, provides a novel combined treatment scheme for clinical treatment of DLBCL, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to application of a pharmaceutical composition in the treatment of diffuse large B-cell lymphoma. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Diffuse large B-cell lymphoma (DLBCL) is a malignant tumor of the hematopoietic system, accounting for approximately 30% of NHL. It has a high incidence, high clinical and biological heterogeneity, and strong invasiveness, seriously threatening the life and health of patients.

[0004] In recent years, with the deepening understanding of the molecular mechanisms of DLBCL development and progression, and the development and clinical application of novel immunotherapies such as cellular immunotherapy and immune checkpoint therapy, the prognosis of DLBCL patients has significantly improved. However, 40% of patients still experience initial treatment resistance or refractory relapse, ultimately leading to death. Therefore, personalized treatment options are urgently needed to improve the prognosis and survival benefits of DLBCL patients. Therefore, the search for new and highly effective anti-tumor drugs to enhance the clinical efficacy of DLBCL patients is of great significance. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention provides the use of pharmaceutical compositions for the treatment of diffuse large B-cell lymphoma. Specifically, the present invention has discovered two pharmaceutical compositions: magnolin and venetoclax, and masitinib and gemcitabine hydrochloride. These compositions have been shown to synergistically inhibit the growth of DLBCL cells, providing a novel combination therapy for the clinical treatment of DLBCL and promising promising clinical applications. Based on these research findings, the present invention has been completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] The first aspect of the present invention provides use of a pharmaceutical composition in preparing a drug for treating diffuse large B-cell lymphoma.

[0008] In the present invention, the pharmaceutical composition is any one of the following groups:

[0009] (a1) magnolin and venetoclax;

[0010] (a2) Masitinib and gemcitabine hydrochloride.

[0011] Wherein, the molar ratio of magnolin to venetoclax is 40-120:0.2-0.8;

[0012] The molar ratio of masitinib to gemcitabine hydrochloride is 1-10:1-10.

[0013] Both of the above-mentioned pharmaceutical compositions can be used to treat diffuse large B-cell lymphoma. Furthermore, according to the present invention, the concept of "treatment" refers to any measure applicable to the treatment of tumors and related diseases, or preventive treatment of such diseases or symptoms, or the prevention of recurrence of such diseases, such as recurrence after the completion of treatment or the treatment of symptoms of an already existing disease, or preemptive intervention to prevent, inhibit, or reduce the occurrence of such diseases or symptoms.

[0014] The second aspect of the present invention provides the use of the above-mentioned pharmaceutical composition in any one or more of the following:

[0015] (b1) Inhibiting the viability of diffuse large B-cell lymphoma cells or preparing products for inhibiting the viability of diffuse large B-cell lymphoma cells;

[0016] (b2) inhibiting the proliferation of diffuse large B-cell lymphoma cells or preparing products for inhibiting the proliferation of diffuse large B-cell lymphoma cells;

[0017] (b3) Inducing diffuse large B-cell lymphoma cell cycle arrest at the G0 / 1 phase (or G2 / M phase) or preparing a product that induces diffuse large B-cell lymphoma cell cycle arrest at the G0 / 1 phase (or G2 / M phase);

[0018] (b4) reducing the mitochondrial membrane potential level in diffuse large B-cell lymphoma cells, increasing the mitochondrial reactive oxygen species level, and promoting mitophagy in diffuse large B-cell lymphoma cells, or preparing a product that reduces the mitochondrial membrane potential level in diffuse large B-cell lymphoma cells, increasing the mitochondrial reactive oxygen species level, and promoting mitophagy in diffuse large B-cell lymphoma cells;

[0019] (b5) Inducing DNA damage in diffuse large B-cell lymphoma cells or preparing a product that induces DNA damage in diffuse large B-cell lymphoma cells.

[0020] The third aspect of the present invention provides a pharmaceutical composition, the active ingredients of which include any one of the following groups:

[0021] (a1) magnolin and venetoclax;

[0022] (a2) Masitinib and gemcitabine hydrochloride.

[0023] Wherein, the molar ratio of magnolin to venetoclax is 40-120:0.2-0.8;

[0024] The molar ratio of masitinib to gemcitabine hydrochloride is 1-10:1-10.

[0025] The pharmaceutical composition has a synergistic effect of resisting diffuse large B-cell lymphoma.

[0026] A fourth aspect of the present invention provides a method for treating diffuse large B-cell lymphoma, comprising: administering the above-mentioned pharmaceutical composition to a subject.

[0027] Beneficial technical effects of one or more of the above technical solutions:

[0028] This technical solution reports for the first time two drug combinations. Specifically, magnolinone can exert its anti-DLBCL effects by regulating cell activity, proliferation, and cell cycle. In vitro experimental results showed that magnolinone significantly reduced the mitochondrial membrane potential of DLBCL cells, increased mitochondrial reactive oxygen species (ROS) levels, and induced PINK1 / Parkin-mediated mitophagy. Furthermore, magnolinone and venetoclax synergistically inhibited DLBCL cell growth.

[0029] Masitinib exerts its anti-DLBCL effects by regulating cell proliferation and cell cycle. Furthermore, masitinib can activate mitophagy mediated by the PINK1-Parkin signaling pathway by increasing mitochondrial reactive oxygen species (ROS). Combining masitinib with gemcitabine hydrochloride synergistically inhibits DLBCL growth by inducing DNA damage.

[0030] In summary, the above technical solution emphasizes the anti-tumor effect of the two groups of drug compositions, provides new ideas for constructing new combination drug strategies, is of great significance for optimizing DLBCL treatment plans and improving patient prognosis, and has good value for practical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 Schematic diagram and molecular formula of the molecular structure of magnolin (A) and venetoclax (B) in Example 1 of the present invention.

[0033] Figure 2 The magnolin in Example 1 of the present invention can inhibit the viability of DLBCL cells in a concentration-dependent manner.

[0034] Figure 3 The magnolin in Example 1 of the present invention can inhibit the proliferation of DLBCL cells in a time-dependent and concentration-dependent manner.

[0035] Figure 4 The magnolin in Example 1 of the present invention induces cell cycle arrest at the G0 / 1 phase in a concentration-dependent manner.

[0036] Figure 5 The magnolin in Example 1 of the present invention reduces the mitochondrial membrane potential level in DLBCL.

[0037] Figure 6 The magnolin in Example 1 of the present invention increases the level of mitochondrial reactive oxygen species in DLBCL.

[0038] Figure 7 This is the result of Example 1 of the present invention showing that magnolianes promote mitophagy in DLBCL. In Figure A, after magnolianes treatment, the colocalization of the mitochondrial protein TOM20 and the autophagosome marker LC3B increased. In Figure B, after magnolianes treatment, the ratio of the autophagy marker LC3B II / I was greater than 1, the expression level of the autophagy-related protein P62 was significantly reduced, and the expression levels of the mitophagy-related proteins PINK1 and Parkin were significantly increased.

[0039] Figure 8 This is the synergistic anti-lymphoma effect of magnolin and venetoclax in Example 1 of the present invention.

[0040] Figure 9 Schematic diagram and molecular formula of the molecular structure of masitinib (A) and gemcitabine hydrochloride (B) in Example 2 of the present invention.

[0041] Figure 10 In Example 2 of the present invention, masitinib can inhibit the proliferation of DLBCL cells in a time-dependent and concentration-dependent manner.

[0042] Figure 11 In Example 2 of the present invention, masitinib induces cell cycle arrest of DLBCL at the G2 / M phase in a concentration-dependent manner.

[0043] Figure 12 Masitinib in Example 2 of the present invention promotes the production of mitochondrial reactive oxygen species in DLBCL.

[0044] Figure 13 This is the mitochondrial autophagy induced by masitinib in DLBCL in Example 2 of the present invention. After masitinib treatment, the expression levels of mitochondrial markers TOM20 and HSP60 proteins were significantly reduced, the expression levels of autophagy-related proteins P62 and LC3B I / II were significantly reduced, and the expression levels of mitophagy-related proteins PINK1 and Parkin were significantly increased.

[0045] Figure 14 This is the synergistic anti-lymphoma effect of masitinib and gemcitabine hydrochloride in Example 2 of the present invention.

[0046] Figure 15 This is the DNA damage in DLBCL induced by masitinib combined with gemcitabine hydrochloride in Example 2 of the present invention; A is the Western Blot result, and B is the immunofluorescence result. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0048] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0049] In a typical embodiment of the present invention, a use of a pharmaceutical composition in preparing a drug for treating diffuse large B-cell lymphoma is provided.

[0050] In the present invention, the pharmaceutical composition is any one of the following groups:

[0051] (a1) magnolin and venetoclax;

[0052] (a2) Masitinib and gemcitabine hydrochloride.

[0053] Wherein, the molar ratio of magnolin to venetoclax is 40-120:0.2-0.8;

[0054] The molar ratio of masitinib to gemcitabine hydrochloride is 1-10:1-10.

[0055] Specifically, the present invention discovered that magnoliarin can exert its anti-DLBCL effects by regulating cell activity, proliferation, and cell cycle. In vitro experimental results showed that magnoliarin significantly reduced the mitochondrial membrane potential of DLBCL cells, increased mitochondrial reactive oxygen species (ROS), and induced PINK1 / Parkin-mediated mitophagy. Furthermore, magnoliarin and venetoclax synergistically inhibited DLBCL cell growth.

[0056] Secondly, the present invention discovered that masitinib exerts anti-DLBCL effects by regulating cell proliferation and cell cycle. Furthermore, masitinib can activate mitophagy mediated by the PINK1-Parkin signaling pathway by increasing mitochondrial reactive oxygen species (ROS). The combination of masitinib and gemcitabine hydrochloride synergistically inhibits DLBCL growth by inducing DNA damage.

[0057] Therefore, both groups of pharmaceutical compositions described above can be used to treat diffuse large B-cell lymphoma. Furthermore, according to the present invention, the concept of "treatment" refers to any measure applicable to the treatment of tumors and related diseases, or preventive treatment of such diseases or symptoms, or the prevention of recurrence of such diseases, such as recurrence after the completion of treatment or the treatment of symptoms of an already existing disease, or preemptive intervention to prevent, inhibit, or reduce the occurrence of such diseases or symptoms.

[0058] In another embodiment of the present invention, the use of the above-mentioned pharmaceutical composition in any one or more of the following is provided:

[0059] (b1) Inhibiting the viability of diffuse large B-cell lymphoma cells or preparing products for inhibiting the viability of diffuse large B-cell lymphoma cells;

[0060] (b2) inhibiting the proliferation of diffuse large B-cell lymphoma cells or preparing products for inhibiting the proliferation of diffuse large B-cell lymphoma cells;

[0061] (b3) Inducing diffuse large B-cell lymphoma cell cycle arrest at the G0 / 1 phase (or G2 / M phase) or preparing a product that induces diffuse large B-cell lymphoma cell cycle arrest at the G0 / 1 phase (or G2 / M phase);

[0062] (b4) reducing the mitochondrial membrane potential level in diffuse large B-cell lymphoma cells, increasing the mitochondrial reactive oxygen species level, and promoting mitophagy in diffuse large B-cell lymphoma cells, or preparing a product that reduces the mitochondrial membrane potential level in diffuse large B-cell lymphoma cells, increasing the mitochondrial reactive oxygen species level, and promoting mitophagy in diffuse large B-cell lymphoma cells;

[0063] (b5) Inducing DNA damage in diffuse large B-cell lymphoma cells or preparing a product that induces DNA damage in diffuse large B-cell lymphoma cells.

[0064] In (b1)-(b5), the product can be a drug or a test reagent for non-medical use. The test reagent can be used for basic research to construct relevant tumor cell or animal models, and then study related mechanisms such as tumor occurrence and development mediated by mitochondrial autophagy in tumor cells.

[0065] When the product is a medicine, the medicine further comprises at least one non-medicinal active ingredient. The non-medicinal active ingredient comprises a pharmaceutically acceptable carrier, excipient and / or diluent.

[0066] In another embodiment of the present invention, a pharmaceutical composition is provided, wherein the active ingredients comprise any one of the following groups:

[0067] (a1) magnolin and venetoclax;

[0068] (a2) Masitinib and gemcitabine hydrochloride.

[0069] Wherein, the molar ratio of magnolin to venetoclax is 40-120:0.2-0.8;

[0070] The molar ratio of masitinib to gemcitabine hydrochloride is 1-10:1-10.

[0071] The pharmaceutical composition has a synergistic effect of resisting diffuse large B-cell lymphoma.

[0072] In another embodiment of the present invention, the pharmaceutical composition further comprises at least one non-pharmaceutical active ingredient, which includes a pharmaceutically acceptable carrier, excipient, and / or diluent.

[0073] In another embodiment of the present invention, the non-drug active ingredients include:

[0074] Pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids; pharmacologically harmless salts (preferably sodium chloride), flavorings, vitamins (preferably vitamin A or vitamin E, tocopherols or provitamins), antioxidants (preferably ascorbic acid), and stabilizers and / or preservatives.

[0075] The dosage forms of the pharmaceutical preparation include liquid dosage forms, solid dosage forms, external preparations and sprays;

[0076] In another specific embodiment of the present invention, the following dosage forms are included: true solutions, colloids, microparticle dosage forms, emulsion dosage forms, mixed dosage forms, tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, inclusion compounds, implants, patches, and ointments.

[0077] In another embodiment of the present invention, a method for treating diffuse large B-cell lymphoma is provided, comprising: administering the above-mentioned pharmaceutical composition to a subject.

[0078] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, most preferably a human.

[0079] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0080] Example 1

[0081] Experimental methods

[0082] 1. Cell Culture

[0083] The human DLBCL cell lines OCI-LY1 and OCI-LY10 used in this study were purchased from ATCC.

[0084] The cells were cultured in IMDM complete medium containing 10% fetal bovine serum in a 37°C cell culture incubator containing 5% CO2. The medium was replaced and passaged every 2-3 days, and cells with better growth status were selected for subsequent experiments.

[0085] 2. Cell counting kit-8 (CCK-8) was used to detect the proliferation level of DLBCL cells

[0086] DLBCL cells in the logarithmic growth phase and in good condition were collected, the supernatant was removed after centrifugation, the cells were resuspended in IMDM complete medium and counted under a microscope, and 5×10 cells were added per well. 4 DLBCL cells were seeded into a 96-well cell culture plate at a concentration of 10 μL / 90 μL. 10 μL of drug at different concentrations was added according to the concentration conversion formula. Blank wells and three replicate wells were set up and gently shaken to mix. The 96-well plate was then placed in a 37°C cell culture incubator with 5% CO2 and cultured. After 24-48 hours, 10 μL of CCK8 reagent was added to each well, taking care to avoid bubbles. After incubation at 37°C in the dark for 1-4 hours, the absorbance (OD) of each well at 450 nm was measured using a microplate reader. A growth curve of DLBCL cells was plotted based on the test results.

[0087] 3. CellTiter-Glo (CTG) assay for DLBCL cell viability

[0088] DLBCL cells in the logarithmic growth phase and in good condition were collected, and the supernatant was removed after centrifugation; the cells were resuspended in IMDM complete medium and counted under a microscope, with 10 cells per well. 4DLBCL cells were seeded into a dark-protected 384-well cell culture plate at a concentration of 1 μL / 24 μL. 1 μL of drug at different concentrations was added according to the concentration conversion formula. Blank wells and three replicate wells were set up and gently shaken to mix. The 384-well plate was placed in a 37°C cell culture incubator containing 5% CO2 and continued to be cultured. After 48 hours, 25 μL of CTG reagent was added to each well, taking care not to generate bubbles. The plates were shaken in the dark on a horizontal shaker for 2 minutes. The plates were incubated in the dark at room temperature for 10 minutes. The fluorescence signal was recorded using a microplate fluorescence detector.

[0089] 4. Flow cytometry detection of DLBCL cell cycle

[0090] DLBCL cells in the logarithmic growth phase and in good growth condition (drug-addition group and control group) were collected and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; the cells were resuspended with pre-cooled sterile PBS solution, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded, and repeated twice; the cells were resuspended with 500 μL pre-cooled sterile PBS solution, and the cell suspension was added dropwise to an EP tube containing 1200 μL pre-cooled anhydrous ethanol, and carefully pipetted to mix; after 24 hours, the EP tube was removed, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded; the cells were resuspended with pre-cooled sterile PBS solution, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded, and repeated twice; after cell counting, the cell amount was adjusted to 1×10 6 For each cell / group, add 500 μL PI / RNase staining buffer to resuspend the cells; incubate in the dark at room temperature for 15-30 minutes; filter the cell suspension with a 400-mesh filter, transfer the cells to a labeled flow cytometer, and monitor the cell cycle.

[0091] 5. Protein Extraction and Western Blotting

[0092] After treatment, DLBCL cells were collected, resuspended, and washed three times in pre-chilled PBS. The supernatant was removed by centrifugation and the appropriate amount (40-150 μL) of protein lysis buffer (RIPA:PMSF:phosphatase inhibitor, volume ratio: 100:1:1) was added, depending on the size of the cell pellets. The cells were vortexed and incubated on ice for 30 minutes. After centrifugation at 12,000 rpm at 4°C for 30 minutes, the supernatant containing the protein was aspirated. Protein concentration was determined using the BCA assay. The supernatant was then mixed with loading buffer at a ratio of 3:1 by volume. The cells were denatured by incubation at 100°C in a metal bath for 10 minutes and stored at -80°C until further use. A 7.5-12% electrophoresis gel was prepared based on the molecular weight of the target protein. The gel was placed in an electrophoresis tank and the appropriate amount of running buffer was added. 40 μg of protein or 4 μL of protein marker was added to each well. Electrophoresis was performed at a constant voltage of 200 V. The electrophoresis time was determined based on the position of the protein marker and bromophenol blue. Cut a PVDF membrane to the size of the gel in advance and soak it in methanol for 1-2 minutes. Place the cut gel, PVDF membrane, and transfer filter paper in transfer buffer and arrange them from bottom to top: transfer filter paper - PVDF membrane - gel - transfer filter paper. Carefully remove any bubbles and place the membrane in a semi-dry transfer tank. Transfer the membrane at 10V for 30 minutes. Block the PVDF membrane in 10% skim milk in TBST for 1-2 hours at room temperature. Then, incubate the membrane in a primary antibody solution of appropriate concentration prepared in primary antibody diluent at 4°C on a shaker overnight. Wash the PVDF membrane three times with TBST and incubate it in a secondary antibody solution in 10% skim milk at room temperature for 1 hour. Wash the membrane again and add a pre-prepared developer (Solution A:Solution B = 1:1). Image the membrane using an Amersham Imager 600 gel imaging instrument. Analyze the grayscale values ​​of the bands using Image J software to assess the expression level of the target molecule.

[0093] 6. Immunofluorescence Staining

[0094] DLBCL cells in the logarithmic growth phase and in good growth condition were collected and divided into 2×10 6 Cells / 2 mL were inoculated into a 6-well cell culture plate, and the diluted magnolia oleate solution was added. A control well was set up. The cell culture plate was placed in a 37°C cell culture incubator containing 5% CO2. After 48 hours, the supernatant was discarded by centrifugation. The cells were washed with pre-cooled sterile PBS solution twice. The cells were resuspended in pre-cooled sterile PBS solution and the cells were counted under a microscope to adjust the cell concentration to 1×10 6 / mL; add about 1mL 4% paraformaldehyde, incubate at room temperature for 15 minutes; wash the cells with pre-cooled sterile PBS solution, repeat three times; add 0.2% TritonX-100, incubate at room temperature for 10 minutes; wash the cells with pre-cooled sterile PBS solution, repeat three times; take 500μL of cell suspension and add it to the circular aperture of the slide, incubate in a 37℃ incubator for 60 minutes; add 2-3 drops of prepared primary antibody solution, place the slide in a humidified box, and incubate overnight at 4℃; immerse the slide in PBS solution for 5 minutes, repeat three times; dry the water around the cells on the slide, add 2-3 drops of prepared secondary antibody solution, incubate in a 37℃ incubator in the dark for 120 minutes; immerse the slide in PBS solution for 5 minutes, repeat three times; dry the water around the cells on the slide, add 1-2 drops of DAPI anti-fluorescence attenuation mounting medium in the dark; slowly cover with a coverslip to avoid bubbles, and observe using a laser confocal microscope.

[0095] 7. MitoSOX superoxide indicator to detect mitochondrial reactive oxygen species levels in DLBCL cells

[0096] DLBCL cells in the logarithmic growth phase and in good growth condition (drug-treated and control groups) were collected and centrifuged, the supernatant removed, and the cells were washed twice with pre-chilled sterile PBS. 13 μL of DMSO was added to 50 μg of MitoSOX Red Mitochondrial Superoxide Indicator to prepare a 5 mM stock solution, which was then diluted to a 500 nM working solution. 1 mL of the probe working solution was added to fully cover the cells, and the cells were incubated in a 37°C cell culture incubator with 5% CO2 in the dark for 30 minutes. The cells were washed three times with PBS and observed using a laser confocal microscope.

[0097] 8. Mitochondrial membrane potential detection kit (JC-1) to detect the mitochondrial membrane potential level of DLBCL cells

[0098] DLBCL cells in the logarithmic growth phase and in good growth condition (drug-treated group and control group) were collected, centrifuged, and the supernatant was removed. The cells were resuspended in 0.5 ml of cell culture medium, 0.5 ml of JC-1 staining working solution was added, mixed by inversion, and incubated in a 37°C cell culture incubator for 20 minutes; centrifuged at 600g for 3-4 minutes at 4°C, and the supernatant was discarded; the cells were washed twice with JC-1 staining buffer; the cells were resuspended in 1 ml of JC-1 staining buffer, centrifuged at 600g for 3-4 minutes at 4°C, and the supernatant was discarded; the cells were resuspended in 1 ml of JC-1 staining buffer, and observed under an inverted fluorescence microscope.

[0099] Experimental results

[0100] Figure 1The schematic diagram and molecular formula of the molecular structure of magnolin and venetoclax are shown.

[0101] Figure 2 It was shown that magnolin could inhibit the survival of DLBCL cells in a concentration-dependent manner.

[0102] Figure 3 It was shown that magnolin could inhibit the proliferation of DLBCL cells in a time-dependent and concentration-dependent manner.

[0103] Figure 4 The results showed that magnolin induced cell cycle arrest at G0 / 1 phase in a concentration-dependent manner.

[0104] Figure 5 It was shown that magnolin reduces mitochondrial membrane potential levels in DLBCL.

[0105] Figure 6 showed that magnolin increased mitochondrial reactive oxygen species levels in DLBCL.

[0106] Figure 7 Magnoliopsin was shown to promote mitophagy in DLBCL. Figure 7 In A, colocalization of the mitochondrial protein TOM20 and the autophagosome marker LC3B increased after magnolin treatment. Figure 7 In Figure B, after treatment with magnoliarin, the expression levels of autophagy-related proteins P62 and LC3B I / II were significantly decreased, while the expression levels of mitophagy-related proteins PINK1 and Parkin were significantly increased. These results indicate that magnoliarin induces PINK1 / Parkin-mediated mitophagy in DLBCL cells.

[0107] Figure 8 The results showed that magnolin and venetoclax have a synergistic anti-lymphoma effect. The CI values ​​of magnolin and venetoclax at different concentrations were all less than 1, indicating a synergistic effect.

[0108] In summary, this study demonstrates for the first time that magnolin can exert its anti-DLBCL effects by regulating cell viability, proliferation, and cell cycle. In vitro experimental results show that magnolin can significantly reduce mitochondrial membrane potential in DLBCL cells, increase mitochondrial reactive oxygen species (ROS) levels, and induce PINK1 / Parkin-mediated mitophagy. Furthermore, magnolin and venetoclax synergistically inhibit DLBCL cell growth. This study is expected to provide a novel combination therapy for the clinical treatment of DLBCL and has broad clinical application prospects.

[0109] Example 2

[0110] Experimental methods

[0111] 1. Cell Culture

[0112] The human DLBCL cell lines OCI-LY1 and OCI-LY3 used in this study were purchased from ATCC.

[0113] The cells were cultured in IMDM complete medium containing 10% fetal bovine serum in a 37°C cell culture incubator containing 5% CO2. The medium was replaced and passaged every 2-3 days, and cells with better growth status were selected for subsequent experiments.

[0114] 2. Cell counting kit-8 (CCK-8) was used to detect the proliferation level of DLBCL cells

[0115] DLBCL cells in the logarithmic growth phase and in good condition were collected, the supernatant was removed after centrifugation, the cells were resuspended in IMDM complete medium and counted under a microscope, and 5×10 cells were added per well. 4 DLBCL cells were seeded into a 96-well cell culture plate at a concentration of 10 μL / 90 μL. 10 μL of drug at different concentrations was added according to the concentration conversion formula. Blank wells and three replicate wells were set up and gently shaken to mix. The 96-well plate was then placed in a 37°C cell culture incubator with 5% CO2 and cultured. After 24-48 hours, 10 μL of CCK8 reagent was added to each well, taking care to avoid bubbles. After incubation at 37°C in the dark for 1-4 hours, the absorbance (OD) of each well at 450 nm was measured using a microplate reader. A growth curve of DLBCL cells was plotted based on the test results.

[0116] 3. Flow cytometry detection of DLBCL cell cycle

[0117] DLBCL cells in the logarithmic growth phase and in good growth condition (drug-addition group and control group) were collected and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; the cells were resuspended with pre-cooled sterile PBS solution, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded, and repeated twice; the cells were resuspended with 500 μL pre-cooled sterile PBS solution, and the cell suspension was added dropwise to an EP tube containing 1200 μL pre-cooled anhydrous ethanol, and carefully pipetted to mix; after 24 hours, the EP tube was removed, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded; the cells were resuspended with pre-cooled sterile PBS solution, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded, and repeated twice; after cell counting, the cell amount was adjusted to 1×10 6 For each cell / group, add 500 μL PI / RNase staining buffer to resuspend the cells; incubate in the dark at room temperature for 15-30 minutes; filter the cell suspension with a 400-mesh filter, transfer the cells to a labeled flow cytometer, and monitor the cell cycle.

[0118] 4. Detection of mitochondrial reactive oxygen species levels in DLBCL cells using MitoSOX superoxide indicator

[0119] DLBCL cells in the logarithmic growth phase and in good growth condition (drug-treated and control groups) were collected and centrifuged, the supernatant removed, and the cells were washed twice with pre-chilled sterile PBS. 13 μL of DMSO was added to 50 μg of MitoSOX Red Mitochondrial Superoxide Indicator to prepare a 5 mM stock solution, which was then diluted to a 500 nM working solution. 1 mL of the probe working solution was added to fully cover the cells, and the cells were incubated in a 37°C cell culture incubator with 5% CO2 in the dark for 30 minutes. The cells were washed three times with PBS and observed using a laser confocal microscope.

[0120] 5. Protein Extraction and Western Blotting

[0121] After treatment, DLBCL cells were collected, resuspended, and washed three times in pre-chilled PBS. The supernatant was removed by centrifugation and the appropriate amount (40-150 μL) of protein lysis buffer (RIPA:PMSF:phosphatase inhibitor, volume ratio: 100:1:1) was added, depending on the size of the cell pellets. The cells were vortexed and incubated on ice for 30 minutes. After centrifugation at 12,000 rpm at 4°C for 30 minutes, the supernatant containing the protein was aspirated. Protein concentration was determined using the BCA assay. The supernatant was then mixed with loading buffer at a ratio of 3:1 by volume. The cells were denatured by incubation at 100°C in a metal bath for 10 minutes and stored at -80°C until further use. A 7.5-12% electrophoresis gel was prepared based on the molecular weight of the target protein. The gel was placed in an electrophoresis tank and the appropriate amount of running buffer was added. 40 μg of protein or 4 μL of protein marker was added to each well. Electrophoresis was performed at a constant voltage of 200 V. The electrophoresis time was determined based on the position of the protein marker and bromophenol blue. Cut a PVDF membrane to the size of the gel in advance and soak it in methanol for 1-2 minutes. Place the cut gel, PVDF membrane, and transfer filter paper in transfer buffer and arrange them from bottom to top: transfer filter paper - PVDF membrane - gel - transfer filter paper. Carefully remove any bubbles and place the membrane in a semi-dry transfer tank. Transfer the membrane at 10V for 30 minutes. Block the PVDF membrane in 10% skim milk in TBST for 1-2 hours at room temperature. Then, incubate the membrane in a primary antibody solution of appropriate concentration prepared in primary antibody diluent at 4°C on a shaker overnight. Wash the PVDF membrane three times with TBST and incubate it in a secondary antibody solution in 10% skim milk at room temperature for 1 hour. Wash the membrane again and add a pre-prepared developer (Solution A:Solution B = 1:1). Image the membrane using an Amersham Imager 600 gel imaging instrument. Analyze the grayscale values ​​of the bands using Image J software to assess the expression level of the target molecule.

[0122] 6. Immunofluorescence Staining

[0123] DLBCL cells in the logarithmic growth phase and in good growth condition were collected and divided into 2×10 6 1×10 cells / 2 mL were inoculated into a 6-well cell culture plate, and diluted masitinib and gemcitabine hydrochloride solutions were added. Control wells were set up. The cell culture plate was placed in a cell culture incubator containing 5% CO2. After 48 hours, the supernatant was discarded by centrifugation. The cells were washed with pre-cooled sterile PBS solution twice. The cells were resuspended in pre-cooled sterile PBS solution and the cells were counted under a microscope to adjust the cell concentration to 1×10 6 / mL; add about 1mL 4% paraformaldehyde and incubate at room temperature for 15 minutes; wash the cells with pre-cooled sterile PBS solution, repeat three times; add 0.2% TritonX-100, incubate at room temperature for 10 minutes; wash the cells with pre-cooled sterile PBS solution, repeat three times; take 500μL of cell suspension and add it to the circular aperture of the slide, incubate in a 37℃ incubator for 60 minutes; add 2-3 drops of prepared primary antibody solution, place the slide in a wet box, and incubate overnight at 4℃; immerse the slide in PBS solution for 5 minutes, repeat three times; dry the water around the cells on the slide, add 2-3 drops of prepared secondary antibody solution, incubate in a 37℃ incubator in the dark for 120 minutes; immerse the slide in PBS solution for 5 minutes, repeat three times; dry the water around the cells on the slide, add 1-2 drops of DAPI anti-fluorescence attenuation mounting medium in the dark; slowly cover with a coverslip to avoid bubbles, and observe using a laser confocal microscope.

[0124] Experimental results

[0125] Figure 9 Schematic diagrams and molecular formulas of the molecular structures of masitinib and gemcitabine hydrochloride are shown.

[0126] Figure 10 It was shown that masitinib could inhibit the proliferation of DLBCL cells in a time-dependent and concentration-dependent manner.

[0127] Figure 11 It was shown that masitinib induced DLBCL cell cycle arrest at the G2 / M phase in a concentration-dependent manner.

[0128] Figure 12 Masitinib was shown to promote the production of mitochondrial reactive oxygen species in DLBCL.

[0129] Figure 13Masitinib-induced mitophagy in DLBCL cells. Masitinib treatment significantly reduced the expression of mitochondrial markers TOM20 and HSP60, increased the ratio of autophagy marker LC3B II / I to greater than 1, and significantly decreased the expression of autophagy-related protein P62. Furthermore, the expression of mitophagy-related proteins PINK1 and Parkin was significantly increased.

[0130] Figure 14 The results showed a synergistic anti-lymphoma effect of masitinib and gemcitabine hydrochloride. The CI values ​​of masitinib and gemcitabine hydrochloride at different concentrations were all less than 1, indicating a synergistic effect.

[0131] Figure 15 The study showed that masitinib combined with gemcitabine hydrochloride induced DNA damage in DLBCL. Masitinib and gemcitabine hydrochloride increased the expression of p-H2AX, a DNA damage marker, in DLBCL cells, and the combination had a synergistic effect.

[0132] This study demonstrates for the first time that masitinib can exert its anti-DLBCL effects by regulating cell proliferation and cell cycle. Furthermore, masitinib can activate mitophagy mediated by the PINK1-Parkin signaling pathway by increasing mitochondrial reactive oxygen species (ROS). The combination of masitinib and gemcitabine hydrochloride synergistically inhibits DLBCL growth by inducing DNA damage. This study highlights the anti-tumor effects of the masitinib and gemcitabine hydrochloride combination, providing new insights into novel combination drug strategies and potentially significant for optimizing DLBCL treatment regimens and improving patient outcomes.

[0133] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of the pharmaceutical composition in the preparation of a drug for the treatment of diffuse large B-cell lymphoma; The pharmaceutical composition is any one of the following groups: (a1) magnolin and venetoclax; (a2) Masitinib and gemcitabine hydrochloride.

2. The use according to claim 1, characterized in that The molar ratio of magnolin to venetoclax is 40-120:0.2-0.8; The molar ratio of masitinib to gemcitabine hydrochloride is 1-10:1-10.

3. Use of the pharmaceutical composition in any one or more of the following: (b1) Inhibiting the viability of diffuse large B-cell lymphoma cells or preparing products for inhibiting the viability of diffuse large B-cell lymphoma cells; (b2) inhibiting the proliferation of diffuse large B-cell lymphoma cells or preparing products for inhibiting the proliferation of diffuse large B-cell lymphoma cells; (b3) Inducing diffuse large B-cell lymphoma cell cycle arrest at the G0 / 1 phase (or G2 / M phase) or preparing a product that induces diffuse large B-cell lymphoma cell cycle arrest at the G0 / 1 phase (or G2 / M phase); (b4) reducing the mitochondrial membrane potential level in diffuse large B-cell lymphoma cells, increasing the mitochondrial reactive oxygen species level, and promoting mitophagy in diffuse large B-cell lymphoma cells, or preparing a product that reduces the mitochondrial membrane potential level in diffuse large B-cell lymphoma cells, increasing the mitochondrial reactive oxygen species level, and promoting mitophagy in diffuse large B-cell lymphoma cells; (b5) inducing DNA damage in diffuse large B-cell lymphoma cells or preparing a product that induces DNA damage in diffuse large B-cell lymphoma cells; The pharmaceutical composition is any one of the following groups: (a1) magnolin and venetoclax; (a2) Masitinib and gemcitabine hydrochloride.

4. The use according to claim 3, characterized in that The molar ratio of magnolin to venetoclax is 40-120:0.2-0.8; The molar ratio of masitinib to gemcitabine hydrochloride is 1-10:1-10.

5. The use according to claim 3, characterized in that The product is a drug or a test reagent for non-medical use, and the test reagent is used for basic research to construct relevant tumor cell or animal models.

6. The use according to claim 3, characterized in that When the product is a medicine, the medicine further comprises at least one non-medicinal active ingredient.

7. The use according to claim 6, characterized in that The non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients and / or diluents.

8. A pharmaceutical composition, characterized in that Its active ingredients include any of the following groups: (a) Magnoliopsin and Venetoclax; (b) Masitinib and gemcitabine hydrochloride.

9. The pharmaceutical composition according to claim 8, wherein The molar ratio of magnolin to venetoclax is 40-120:0.2-0.8; The molar ratio of masitinib to gemcitabine hydrochloride is 1-10:1-10.

10. The pharmaceutical composition according to any one of claims 8 to 9, characterized in that The pharmaceutical composition has a synergistic effect of resisting diffuse large B-cell lymphoma.

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