Schisandrin A in the preparation of drugs for treating multiple myeloma

Abstract

This invention relates to the field of biomedical technology, specifically to the application of schisandrin A in the preparation of drugs for treating multiple myeloma. The schisandrin A (SchA) provided by this invention exerts its therapeutic effect on multiple myeloma by synergistically inducing apoptosis and mitophagy in multiple myeloma cells. The structural formula of the schisandrin A is as follows:

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of schisandrin A in the preparation of drugs for treating multiple myeloma. Background Technology

[0002] Multiple myeloma (MM) is the second most common hematologic malignancy, characterized by the malignant proliferation of plasma cells in the bone marrow. Although the use of proteasome inhibitors (such as bortezomib), immunomodulators, and monoclonal antibodies has improved patient prognosis, the vast majority of patients eventually develop drug resistance and relapse, leading to treatment failure. Therefore, developing new anti-MM drugs with unique mechanisms of action is crucial. Long-term use of existing mainstream chemotherapy drugs and targeted therapies easily leads to acquired resistance in MM cells. Some drugs have significant neurotoxicity, myelosuppression, and other side effects, impacting patients' quality of life.

[0003] Mitochondrial autophagy and apoptosis are two important modes of programmed cell death, playing complex roles in tumorigenesis and development. Most currently used anti-MM drugs rely primarily on inducing apoptosis as their anti-tumor mechanism. For example, the second-generation proteasome inhibitor carfilzomib inhibits the ubiquitin-proteasome pathway, inducing endoplasmic reticulum stress and activating endogenous and exogenous caspase pathways, ultimately exerting its anti-MM effect mainly through inducing tumor cell apoptosis. The immunomodulator lenalidomide recruits transcription factors IKZF1 and IKZF3 to cereblon (CRBN) for ubiquitination and degradation, thereby inhibiting MM cell proliferation, but its core mechanism is also primarily apoptosis induction. Drugs capable of precisely and synergistically regulating these two pathways for therapeutic effect are few. Therefore, it is necessary to develop new approaches to synergistically treat multiple myeloma by co-regulating the mitochondrial autophagy and apoptosis pathways. Summary of the Invention

[0004] To develop a therapeutic approach for multiple myeloma by synergistically regulating the mitophagy and apoptosis pathways, this invention provides the application of schisandrin A in the preparation of drugs for treating multiple myeloma. The schisandrin A (SchA) provided by this invention exerts its therapeutic effect on multiple myeloma by synergistically inducing apoptosis and mitophagy in multiple myeloma cells.

[0005] This invention provides the application of schisandrin A in the preparation of drugs for treating multiple myeloma, wherein the molecular formula of schisandrin A is C0. 24 H 32 O6, chemical structural formula as follows: .

[0006] Furthermore, the active ingredient of the drug is schisandrin A, or a combination of schisandrin A and bortezomib.

[0007] Furthermore, when the active ingredient of the drug is a combination of schisandrin A and bortezomib, the schisandrin A and bortezomib synergistically inhibit the proliferation of human multiple myeloma cells.

[0008] Furthermore, the human multiple myeloma cells include RPMI 8226 and L363.

[0009] Furthermore, the schisandrin A exerts its therapeutic effect on multiple myeloma by synergistically inducing apoptosis and mitophagy in multiple myeloma cells.

[0010] Furthermore, the synergistic induction of apoptosis and mitophagy by schisandrin A is achieved by regulating the "ROS / AMPK / mTOR" signaling axis.

[0011] Furthermore, the regulation of the "ROS / AMPK / mTOR" signaling axis includes: increasing the level of reactive oxygen species in multiple myeloma cells, activating AMP-dependent protein kinases, and inhibiting the activity of mammalian target of rapamycin complex 1.

[0012] Furthermore, the induction of apoptosis includes: upregulating the expression of the pro-apoptotic protein Bax, downregulating the expression of the anti-apoptotic protein Bcl-2, and activating Caspase-3; The induced mitophagy includes: upregulating the expression of PINK1 and Parkin proteins, promoting the conversion of LC3-II protein, and degrading p62 protein.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: Schisandrin A (SchA) of this invention is used to prepare drugs for the prevention and / or treatment of multiple myeloma. Schisandrin A exerts its anti-multiple myeloma effect through the following integration mechanism:

[0014] (1) Initiation signal: SchA first targets and damages the mitochondria of tumor cells, leading to a burst of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential.

[0015] (2) Activation of core pathways: Elevated ROS activates AMP-dependent protein kinase (AMPK) while inhibiting the activity of mammalian target of rapamycin complex 1 (mTORC1), i.e., regulating the “ROS / AMPK / mTOR” signaling axis.

[0016] (3) SchA co-induced dual death procedure: Pathway 1 (apoptosis): AMPK activation and mitochondrial damage work together to upregulate the pro-apoptotic protein Bax and downregulate the anti-apoptotic protein Bcl-2, leading to permeation of the mitochondrial outer membrane, release of cytochrome C, and ultimately activation of Caspase-3 to initiate apoptosis.

[0017] Pathway 2 (autophagy): Inhibition of mTORC1 releases the brakes on autophagy; simultaneously, the PINK1 protein accumulated on damaged mitochondria recruits and activates Parkin, mediating mitochondrial ubiquitination. Ubiquitinated mitochondria bind to the autophagosome membrane protein LC3-II via the aptamer protein p62, are encapsulated to form mitophagosomes, and are eventually degraded in lysosomes (manifested as a decrease in p62 protein levels).

[0018] Synergistic effect: The two pathways mentioned above are not independent, but form a positive feedback loop. Apoptosis exacerbates mitochondrial damage, providing more substrates for mitophagy; while mitophagy effectively clears damaged mitochondria, accelerating cellular energy collapse, thus irreversibly promoting the apoptosis process. This synergistic effect is key to SchA's efficient killing of MM cells.

[0019] The SchA of the present invention can also be used in combination with at least one other anti-multiple myeloma drug (such as the proteasome inhibitor bortezomib) to prepare a combination therapy with synergistic effects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The effect of different SchA concentrations on the viability of multiple myeloma cells; In the figure, A represents the cell viability of the human multiple myeloma cell line RPMI 8226 after 24 hours of treatment with different concentrations of SchA; B represents the IC50 values ​​of different concentrations of SchA used to treat the human multiple myeloma cell line RPMI 8226. C represents the cell viability of human multiple myeloma cell line L363 after 24 hours of treatment with different concentrations of SchA; D represents the IC50 values ​​of human multiple myeloma cell line L363 treated with different concentrations of SchA.

[0022] Figure 2The effect of different concentrations of SchA on the apoptosis rate of human multiple myeloma cell lines RPMI 8226 and L363; In the figure, A represents the apoptosis of human multiple myeloma cell lines RPMI8226 and L363 by different concentrations of SchA detected by flow cytometry. B is a statistical graph showing the apoptosis rate of different concentrations of SchA on human multiple myeloma cell lines RPMI 8226 (left) and L363 (right).

[0023] Figure 3 SchA dose-dependently regulates the expression of key proteins in the apoptosis pathway of multiple myeloma cells; In the figure, A represents the dose-dependent regulation of the expression of key proteins in the RPMI 8226 apoptosis pathway in multiple myeloma cells by SchA; B is a dose-dependent regulator of the expression of key proteins in the L363 apoptosis pathway in multiple myeloma cells by SchA.

[0024] Figure 4 SchA activates the AMPK / mTOR axis and induces autophagy. In the figure, A represents the situation where SchA activates the AMPK / mTOR axis and induces autophagy in L363 multiple myeloma cells; B represents the scenario where SchA activates the AMPK / mTOR axis and induces autophagy in multiple myeloma cells RPMI 8226.

[0025] Figure 5 Synergistic spectrum of schisandrin A combined with bortezomib; In the figure, A is a two-dimensional heatmap of SchA and bortezomib in the multiple myeloma cell line RPMI 8226, showing the synergistic distribution of different concentration combinations and ZIP scores; B is a three-dimensional surface plot of SchA combined with bortezomib in the multiple myeloma cell line RPMI 8226, showing the synergistic peak and overall trend at different concentrations; C is a two-dimensional heatmap of SchA and bortezomib in the L363 multiple myeloma cell line, showing the synergistic distribution of different concentration combinations and ZIP scores; D is a three-dimensional surface plot of SchA combined with bortezomib in the L363 multiple myeloma cell line, showing the synergistic peak and overall trend at different concentrations.

[0026] Figure 6 To detect the apoptosis rate of the combination of schisandrin A and bortezomib. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0028] Example 1: Application of schisandrin A in the preparation of drugs for treating multiple myeloma.

[0029] I. Experimental Materials and Methods 1. Experimental materials The schizandrin A (CAS No.: 61281-38-7) used in this invention was purchased from MedChemExpress, USA. Its chemical name is Schizandrin-A; Wuweizisu-A; Deoxyschizandrin, and its purity, as determined by liquid chromatography-mass spectrometry (LCMS), reached 99.89%.

[0030] Preparation of Schisandrin A: According to the product instructions, schisandrin A is dissolved in dimethylsulfoxide (DMSO) and then sonicated to promote complete dissolution. A stock solution with a final concentration of 100 mM is then prepared, aliquoted, and stored at -80°C for later use. During the experiment, the stock solution is diluted with cell culture medium to the required working concentration according to specific experimental needs.

[0031] 2. Cell Culture Cell resuscitation: Cell resuscitation is a crucial step in restoring the viability of cryopreserved cells. First, all necessary reagents and consumables are placed in a laminar flow hood and sterilized under UV light for 30 minutes, with laminar flow ventilation to ensure a sterile environment. Then, the cryopreserved cell tubes are removed from the liquid nitrogen container and quickly placed in a 37°C water bath, gently agitated clockwise to thaw rapidly. Once thawed, the cells are immediately transferred to the laminar flow hood and added to a 15ml centrifuge tube containing 10ml of complete culture medium, gently mixed by pipetting. Next, centrifuge at 1000rpm for 5 minutes, discard the supernatant, gently resuspend the cell pellet in 5ml of complete culture medium, and transfer it to a cell culture flask. Finally, label the cell culture flask with the cell name and resuscitation date, and place the flask in a 37°C, 5% CO2 cell culture incubator for further culture.

[0032] Cell passage: Cell passage is used to maintain cell growth and increase cell numbers. First, place all necessary reagents and consumables in a clean bench and sterilize with UV light for 30 minutes, then turn on laminar flow. Observe the cell state under an inverted microscope. When the cells reach the logarithmic growth phase and the density reaches approximately 80%-90% confluence, passage can be performed. Passage is usually performed at a 1:2 ratio. The specific procedure is as follows: Transfer the cell suspension to a 15ml sterile centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend the cells in fresh complete culture medium, and gently pipette to mix. Finally, evenly distribute the cell suspension into two new culture flasks, label them with the date and passage number, and place the cell culture flasks in a cell culture incubator for continued culture.

[0033] Cell cryopreservation: Cell cryopreservation is used for long-term preservation of cell lines to ensure the stability of their biological characteristics. First, place the necessary experimental reagents and consumables in a clean bench for UV sterilization for 30 minutes, and turn on the laminar flow. Observe the cell state under an inverted microscope and select cells in the logarithmic growth phase and in optimal condition for cryopreservation. The specific procedure is as follows: Transfer the cell suspension to a 15ml sterile centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and add an appropriate amount of cell cryopreservation solution according to the cell count (generally per 1×10⁻⁶ cells). 7 Add 1 ml of cell freezing solution to each cell and gently pipette to mix. Transfer the cell suspension to cryovials. Clearly label the cryovials with the cell name and freezing date. Then, place the cryovials in a -80°C freezer overnight, and transfer them to liquid nitrogen for long-term storage the following day.

[0034] 3. CCK8 assay for cell viability (1) Cell preparation for plating: Select cells in the logarithmic growth phase and collect them into 15 ml sterile centrifuge tubes. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Resuspend the cells in 1 ml of fresh culture medium and count them. Adjust the cell density to ensure that the final volume per well is 100 μl containing 5 × 10⁶ cells / mL. 4 Cells were seeded into 96-well plates, with four replicates per well. The blank control wells contained the same volume of complete culture medium as the experimental wells, without cells.

[0035] (2) Drug intervention: Schisandrin A at different concentrations was added to the cells of the experimental group, with concentration gradients of 0, 10, 50, 100, 200 and 400 μM. After gently mixing, the 96-well plate was placed on a cell shaker to ensure uniform cell distribution, and then placed in a cell culture incubator for culture.

[0036] (3) CCK8 reagent addition: After 0h, 24h, 48h and 72h of schisandrin intervention in cells, remove the 96-well plate and add 10μl of CCK8 solution to each well under light-protected conditions. Care should be taken during the operation to avoid generating air bubbles that could affect the experimental results. After adding CCK8, place the 96-well plate on a cell shaker to mix, and then place it in a cell culture incubator for another 2h.

[0037] (4) Data collection: After incubation, the absorbance (Optical Density, OD) of each well was measured at a wavelength of 450 nm using a multi-functional microplate reader. The experimental data were recorded and statistical analysis was performed.

[0038] 4. Flow cytometry detection of apoptosis (1) Cell preparation and plating: Select cells in the logarithmic growth phase and collect them into 15 ml sterile centrifuge tubes. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Resuspend the cells in an appropriate amount of complete culture medium and adjust the cell density to ensure that 1 × 10⁶ cells are seeded per well. 6 Cells were seeded into 6-well plates, with 3 replicates per group.

[0039] (2) Drug intervention: Schisandrin A at different concentrations was added to the experimental group, with concentration gradients of 0 μM, 80 μM, 120 μM and 160 μM, and the cells were intervened for 24 h.

[0040] (3) Cell collection and washing: After the intervention, the cells were transferred to flow cytometry tubes and collected by centrifugation at 1000 rpm for 5 minutes. The supernatant was discarded. The cells were resuspended in pre-cooled PBS and washed twice by centrifugation at 1000 rpm for 5 minutes to completely remove residual culture medium and drugs.

[0041] (4) Staining treatment: Follow the instructions in the kit, prepare Annexin-binding Buffer with pure water at a ratio of 5:1, and resuspend the cells in it. Then, add 10 μl of Annexin V-fluorescein isothiocyanate (FITC) and 5 μl of propidium iodide (PI) solution to each sample, mix thoroughly, and incubate at room temperature in the dark for 5 minutes.

[0042] (5) Flow cytometry analysis: After incubation, the samples were loaded onto the flow cytometer to detect and analyze the apoptosis rate of all samples.

[0043] 5. Western Blot Detection (1) Protein extraction ① Cell collection and washing: After intervening with different concentrations of schisandrin A (0, 80, 120, 160 μM) for 24 h, cells were collected by centrifugation at 1000 rpm for 5 min and washed twice with pre-chilled PBS. Subsequently, the cells were resuspended in 1 ml of pre-chilled PBS and transferred to 1.5 ml EP tubes. The cells were then centrifuged again at 1000 rpm for 5 min at 4 °C. The supernatant was discarded, and the cell pellet was placed on ice for later use.

[0044] ② Preparation of protein lysis buffer: Based on the amount of cell pellet in step ①, prepare an appropriate amount of protein lysis buffer fresh for use. Mix RIPA lysis buffer: PMSF protease inhibitor: phosphorylase inhibitor in a ratio of 100:1:1, vortex to mix, and place on ice for later use.

[0045] ③ Cell lysis and centrifugation: Add the prepared protein lysis buffer (100 μl) to the 1.5 ml EP container from step ①, and vortex to fully lyse the cells. Place the lysed sample on ice for 20 min, and then centrifuge at 12000 g for 15 min using a cryogenic centrifuge.

[0046] ④ Protein supernatant collection: After centrifugation, carefully transfer the supernatant to a new 1.5ml EP tube that has been pre-chilled and place it on ice for later use.

[0047] (2) Determination of protein concentration by BCA method ① Preparation of standard: Prepare BSA standard by serial dilution to 1 μg / μL, 0.5 μg / μL, 0.25 μg / μL, 0.125 μg / μL, 0.0625 μg / μL, 0.03125 μg / μL and 0 μg / μL blank control. In this setup, tube A contains 100 μL of diluent and 100 μL of BSA standard, resulting in a final concentration of 1 μg / μL. Tube B contains 100 μL of diluent, and 100 μL of the solution from tube A is mixed and added to tube B, resulting in a final concentration of 0.5 μg / μL. Tube C contains 100 μL of diluent, and 100 μL of the solution from tube B is mixed and added to tube C, resulting in a final concentration of 0.25 μg / μL. Tube D contains 100 μL of diluent, and 100 μL of the solution from tube C is mixed and added to tube D, resulting in a final concentration of 0.125 μg / μL. Tube E contains 100 μL of diluent, and 100 μL of the solution from tube D is mixed and added to tube E, resulting in a final concentration of 0.0625 μg / μL. Tube F contains 100 μL of diluent, and 100 μL of the solution from tube E is mixed and added to tube F, resulting in a final concentration of 0.03125 μg / μL. μg / μL; add 100 μL of diluent to tube G, without adding BSA standard, as a blank control. Mix each tube thoroughly during preparation to avoid generating air bubbles.

[0048] ② Preparation of BCA working solution: The volume of BCA working solution per well is 200 μL. Based on the calculated volume, mix BCA-ASolution and BCA-B Solution at a volume ratio of 50:1, mix thoroughly, and prepare the BCA working solution. Prepare and use immediately.

[0049] ③ Sample addition reaction: Take a 96-well microplate and add BSA standard, blank control, and the protein sample to be tested sequentially according to the pre-designed well positions. Add 25 μL of diluted BSA standard or the protein sample to be tested to each well. Set up 3 replicates for each standard and the sample to be tested. If the protein concentration of the sample to be tested is high, the sample can be pre-diluted 5 or 10 times using a diluent consistent with the lysis buffer as needed.

[0050] ④ Reaction and incubation: Add 200 μl of BCA working solution to the standard wells and the sample wells to be tested, mix thoroughly, and incubate in a 37℃ incubator in the dark for 30 min.

[0051] ⑤ Detection and Calculation: The OD value of each well was detected at a wavelength of 562 nm using a multi-functional microplate reader. A standard curve was plotted based on the measured OD values, and the concentration of the protein sample was calculated.

[0052] (3) Preparation of protein samples ① Protein sample loading: Add 2 μl of the protein sample obtained in step (1) to a 96-well plate and dilute it 10 times with PBS. Set up 3 replicates for each protein sample.

[0053] ② BCA Quantitative Verification: Prepare BCA working solution according to the required amount. Add 200 μl of BCA working solution to each well and shake well in a figure-eight motion. Incubate at 37℃ for 30 min. Measure the OD at a wavelength of 562 nm using a multi-mode microplate reader. Calculate the protein concentration after dilution based on the standard curve obtained in step (2).

[0054] ③ Protein denaturation treatment: Mix the protein sample with 5× protein loading buffer at a ratio of 4:1, mix thoroughly by pipetting, boil in a 100℃ metal bath for 10 minutes, then quickly transfer to ice to cool, and finally store in a -20℃ freezer for later use.

[0055] (4) SDS-PAGE gel electrophoresis ① Gel Preparation: Wash and dry the gel preparation frame and glass plates. Clamp the thin and thick glass plates together using the frame and test for leaks with pure water. Select a separating gel of different concentrations according to the target molecular weight and prepare it using a one-step method. Use two glass beakers and add appropriate amounts of separating gel and concentrated acid gel according to the instructions. Add the coagulant before adding the gel and immediately shake thoroughly to mix. Next, quickly pour the lower layer of gel between the glass plates. When adding the upper layer of gel, pour it slowly, then insert a comb with the appropriate number of holes (observe carefully to prevent air bubbles). Let it stand at room temperature for 15 minutes, following the principle of preparing the gel immediately before use.

[0056] ② Sample Loading: Install the glass gel plate on the electrophoresis apparatus. While adding electrophoresis buffer to the inner glass tank, observe for leaks. After leak detection, replenish the electrophoresis buffer, slowly pull out the comb vertically upwards, and use a 200μl pipette to remove any remaining gel from the sample wells. Maintain a protein loading volume of 20μg per well, calculated based on the protein concentration determined by BCA. Add markers and pre-extracted protein samples sequentially according to the experimental groups. Protein samples must be centrifuged before loading. The loading volume can be fine-tuned later based on the observed internal control bands. ③ Electrophoresis: Replenish the inner tank with electrophoresis buffer and add an appropriate amount of electrophoresis buffer to the outer tank. Electrophoresis is performed under constant voltage conditions. The initial electrophoresis voltage is set to 80V for 30 minutes. When the protein sample indicator reaches the interface between the stacking gel and the separating gel, the electrophoresis voltage is adjusted to 120V. When the bromophenol blue indicator reaches the bottom of the gel and the target protein is observed to be separated according to the marker, the electrophoresis is stopped. The process takes approximately 1 hour.

[0057] ④ Transfer: Prepare the transfer sponge and transfer clamps in advance, and soak them in the prepared rapid transfer buffer (rapid transfer buffer: anhydrous ethanol: pure water = 1:1:8). After electrophoresis, transfer the glass gel plate to the transfer buffer, pry open the glass plate, and cut the gel corresponding to the target protein according to the marker indication. Cut the PVDF membrane according to the size of the cut gel. Activate the PVDF membrane in methanol for 30 seconds, then place it in the transfer buffer for later use. Place the transfer clamps in the following order: filter paper-free transfer sponge - PVDF membrane - electrophoresis gel - filter paper-free transfer sponge. Ensure no air bubbles are generated between the PVDF membrane and the electrophoresis gel, and connect the positive and negative electrodes correctly. Use constant current for transfer, with a transfer current of 400mA. The transfer time should be determined according to the molecular weight of the target protein.

[0058] ⑤ Blocking: After the transfer is completed, the PVDF membrane with transferred protein is placed in a pre-prepared TBST solution of 5% skim milk powder and blocked on a shaker at 60 rpm for 90 min at room temperature.

[0059] ⑥ Incubation with primary antibody (target protein-related antibody): After blocking, wash the PVDF membrane with TBST solution for 10 min at 120 rpm on a shaker at room temperature, repeating 3 times. Prepare the primary antibody solution in advance and place it in the antibody incubation box. Place the washed PVDF membrane in the corresponding position of the primary antibody and incubate overnight at 4°C.

[0060] ⑦ Incubation with secondary antibody (goat anti-rabbit IgG-HRP / goat anti-mouse IgG-HRP): On the morning of the first day, recover the primary antibody, wash the PVDF membrane with TBST solution for 10 min at 120 rpm on a shaker at room temperature, repeat 3 times, then add the pre-diluted secondary antibody and incubate at 60 rpm on a shaker at room temperature for 1 h. After recovering the secondary antibody, wash the membrane with TBST solution for 10 min at 120 rpm on a shaker at room temperature, repeat 3 times.

[0061] ⑧ ECL Development: According to the instructions, prepare luminescent solutions A and B in equal proportions, place the PVDF film in the luminescent solution, and expose and develop it using an ultrasensitive multi-functional luminescent imaging device.

[0062] 7. Statistical methods In this invention, experimental data are expressed as mean ± standard deviation (mean ± SD). Qualitative data analysis employed the chi-square test or Fisher's exact test, while quantitative data were compared across multiple groups using one-way ANOVA or nonparametric tests. Differences between two groups were assessed using the independent samples t-test, while comparisons across multiple groups were performed using Analysis of Variance (ANOVA). All statistical analyses and chart generation were performed using GraphPad Prism 10.0 software, with p < 0.05 considered statistically significant.

[0063] II. Experimental Results 1. SchA inhibits MM cell viability and induces apoptosis (in vitro verification) Human multiple myeloma cell lines RPMI 8226 and L363 were treated with different concentrations (0, 10 μM, 50 μM, 100 μM, 200 μM, 400 μM) of SchA for 24 hours. Cell viability was assessed using the CCK-8 assay. Results are shown below. Figure 1 As shown, SchA significantly inhibited the proliferation of both cell types in a dose-dependent manner, and its IC50 value was calculated.

[0064] Cells were treated with 80, 120, and 160 μM SchA, and apoptosis was detected by Annexin V-FITC / PI double staining flow cytometry. The results showed that the proportion of early and late apoptotic cells increased significantly with increasing drug concentration (e.g., at 160 μM, the apoptosis rate of RPMI 8226 cells reached 75.3%). Figure 2As shown in the figure. Western blot analysis revealed that the expression of pro-apoptotic proteins Bax and Cleaved Caspase-3 was upregulated, while the expression of the anti-apoptotic protein Bcl-2 was downregulated. The results are as follows. Figure 3 As shown.

[0065] 2. SchA activates the AMPK / mTOR axis and induces autophagy. The results are as follows Figure 4 As shown, RPMI 8226 cells were treated with an effective concentration of SchA. Western blot analysis showed:

[0066] (1) The level of phosphorylated AMPK (p-AMPK) was significantly increased.

[0067] (2) Phosphorylated mTOR (p-mTOR) was significantly reduced.

[0068] (3) The level of autophagy marker LC3-II was increased, while the level of autophagy substrate p62 protein was decreased.

[0069] (4) The expression of key mitophagy proteins PINK1 and Parkin was upregulated.

[0070] This result confirms that SchA can activate the ROS / AMPK / mTOR signaling axis and induce mitophagy.

[0071] 3. Synergistic effect of SchA and bortezomib (BTZ) To evaluate the combined inhibitory effect of schisandrin A and bortezomib on multiple myeloma cells, this study used L363 cells and RPMI 8226 cells as experimental subjects for combined drug treatment. Based on the cell proliferation inhibition rates obtained from previous single-drug treatments, the corresponding IC50 values ​​for SchA and BTZ were selected respectively. 10 IC 20 and IC 40 The concentration levels were used in subsequent combination drug experiments. Specifically, the IC50 of SchA in L363 cells... 10 IC 20 and IC 40 The IC50 values ​​of BTZ at concentrations of 55 μM, 70 μM, and 100 μM were... 10 IC 20 and IC 40 The concentrations were 5.5 nM, 6.5 nM, and 8.5 nM, respectively; the IC50 of SchA in RPMI 8226 cells was... 10 IC 20 and IC 40 The IC50 values ​​of BTZ at concentrations of 40 μM, 55 μM, and 80 μM were... 10 IC20 and IC 40 The concentrations were 4.5 nM, 5.5 nM, and 7.5 nM, respectively. After combining these different concentrations of SchA with BTZ, the treatments were compared with those of the SchA-only treatment group, the BTZ-only treatment group, and the blank control group. Changes in cell viability were detected in each group, and the combined inhibitory effect and synergistic effect of the two drugs in multiple myeloma cells were further analyzed. This study used SynergyFider software for visualization analysis of the drug combination and calculated the synergistic evaluation based on the Zero Interaction Potency (ZIP) model. The results showed that the ZIP score of RPMI8226 cells was 26.616, and the ZIP score of L363 cells was 22.908. Figure 5 According to the ZIP model criteria, a positive ZIP score indicates a synergistic effect between the drugs, and the higher the score, the more significant the synergistic effect. Therefore, the above results further confirm the synergistic effect of schisandrin A and bortezomib in inhibiting MM cell growth. Flow cytometry was used to further detect the effect of combined treatment with SchA and BTZ on apoptosis of multiple myeloma cells. In the combined drug experiment, SchA and BTZ were used at their respective IC50 values. 20 The concentrations were adjusted. Results showed that the apoptosis rate in the combined treatment group was higher than that in any single-drug treatment group, indicating that the combined application of SchA and BTZ can enhance the induction of apoptosis in multiple myeloma cells. (See results below.) Figure 6 As shown.

[0072] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of schisandrin A in the preparation of drugs for treating multiple myeloma, characterized in that, The structural formula of schisandrin A is as follows: 。 2. The application of schisandrin A according to claim 1 in the preparation of drugs for treating multiple myeloma, characterized in that, The active ingredient of the drug is schisandrin A, or a combination of schisandrin A and bortezomib.

3. The application of schisandrin A according to claim 2 in the preparation of drugs for treating multiple myeloma, characterized in that, When the active ingredient of the drug is a combination of schisandrin A and bortezomib, the schisandrin A and bortezomib synergistically inhibit the proliferation of human multiple myeloma cells.

4. The use of schisandrin A according to claim 3 in the preparation of drugs for treating multiple myeloma, characterized in that, The human multiple myeloma cells mentioned include RPMI 8226 and L363.

5. The use of schisandrin A according to claim 1 in the preparation of drugs for treating multiple myeloma, characterized in that, The schisandrin A exerts its therapeutic effect on multiple myeloma by synergistically inducing apoptosis and mitochondrial autophagy in multiple myeloma cells.

6. The use of schisandrin A according to claim 5 in the preparation of drugs for treating multiple myeloma, characterized in that, The synergistic induction of apoptosis and mitophagy is achieved by regulating the "ROS / AMPK / mTOR" signaling axis.

7. The use of schisandrin A according to claim 6 in the preparation of a drug for treating multiple myeloma, characterized in that, The regulation of the "ROS / AMPK / mTOR" signaling axis includes: increasing the level of reactive oxygen species in multiple myeloma cells, activating AMP-dependent protein kinases, and inhibiting the activity of mammalian target of rapamycin complex 1.

8. The use of schisandrin A according to claim 6 in the preparation of a drug for treating multiple myeloma, characterized in that, The induction of apoptosis includes: upregulating the expression of the pro-apoptotic protein Bax, downregulating the expression of the anti-apoptotic protein Bcl-2, and activating Caspase-3; The induced mitophagy includes: upregulating the expression of PINK1 and Parkin proteins, promoting the conversion of LC3-II protein, and degrading p62 protein.

Images