Application of polyphyllin VII in treatment of myelodysplastic syndrome

Abstract

The invention relates to the technical field of medicines, and provides application of polyphyllin VII in treatment of myelodysplastic syndrome. Furthermore, the invention also provides a medicinal preparation for treating the myelodysplastic syndrome, and the medicinal preparation consists of the polyphyllin VII or the polyphyllin VII salt and pharmaceutically acceptable auxiliary materials. In-vitro pharmacodynamic experiments prove that the polyphyllin VII has a remarkable killing effect on myelodysplastic syndrome tumor cells, and the mechanism of the polyphyllin VII is to promote iron accumulation of the tumor cells and promote lipid peroxidation and oxidative stress so as to induce ferroptosis of the tumor cells. Therefore, a new basis is provided for treating the myelodysplastic syndrome by the polyphyllin VII or the salt thereof, and a new research idea is also provided for treating the myelodysplastic syndrome.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the use of paris saponin VII in preparing a drug for treating myelodysplastic syndrome, and a pharmaceutical preparation containing paris saponin VII or a paris saponin VII salt. Background Art

[0002] Myelodysplastic syndrome is a malignant tumor of the myeloid hematopoietic system. It is a tumor caused by the clonal expansion of multipotent hematopoietic progenitor cells with somatic mutations. It is characterized by abnormal blood cell morphology, blood cell reduction and high risk of progression to acute myeloid leukemia. Myelodysplastic syndrome has great heterogeneity, but eventually progresses to acute myeloid leukemia. Bone marrow transplantation is the only means of curing this disease. Western medicine clinical practice often uses demethylation therapy or combined chemotherapy, which to a certain extent delays the progression of the disease to acute myeloid leukemia, but it cannot cure it. It is accompanied by side effects such as bone marrow suppression and hepato-renal toxicity of chemotherapy drugs, which limits the continued application of chemotherapy. At present, there is a lack of effective drugs for the treatment of myelodysplastic syndrome, so finding drugs for the treatment of myelodysplastic syndrome with definite efficacy, few side effects, and easy acceptance by patients has broad application prospects.

[0003] Compared with general macromolecular drugs, natural small molecule compounds have the characteristics of less side effects, easy absorption and easy to pass through the blood-brain barrier, and a wide range of sources for the treatment of diseases. Paris polyphylla saponin VII (PS VII) is an isospirostanol-type steroidal saponin with a molecular formula of C 51 H 82 O 21 The structural formula is shown in Formula 1, the molecular weight is 1031.18, and it is a white powder. The compound is mainly derived from the leaves and stems of the Paris polyphylla plant and has multiple pharmacological effects such as anti-solid tumor, antibacterial and immunomodulatory.

[0004]

[0005] In terms of the treatment of solid tumors, Chinese patent document CN109331031A discloses the use of Paris saponin VII in drugs for the treatment of breast cancer; Chinese patent document CN106668042A discloses the use of Paris saponin VII in the preparation of anti-lung cancer drugs.

[0006] In terms of myeloma, Chinese patent document CN112402438A discloses the use of Paris saponin VII in the treatment of multiple myeloma, and Chinese patent document CN112426432A discloses the use of Paris saponin VII combined with proteasome inhibitors in the treatment of multiple myeloma. Although both involve malignant changes in the bone marrow, the pathogenesis of the above-mentioned multiple myeloma and the myelodysplastic syndrome involved in the present invention are completely different: multiple myeloma is a plasma cell malignancy originating from the B lymphocyte lineage, with clinical manifestations of hypercalcemia, renal damage, anemia and bone destruction as the main manifestations, and the pathogenesis is complex and often involves abnormal activation of the NF-κB pathway. Clinically, anti-B cell therapy with proteasome inhibitors and CD38 monoclonal antibodies is often used. However, myelodysplastic syndrome is a myeloid malignancy originating from early myeloid stem cells, with different clinical manifestations, often with hematopoiesis as the main symptom, and the pathogenesis is complex, with somatic mutations and abnormal methylation modifications as the main symptoms. Clinically, demethylating drugs such as azacitidine are often used for treatment. Therefore, there has been no report on the application of Paris saponin VII in the treatment of myelodysplastic syndrome. Summary of the invention

[0007] The present invention is made to solve the above-mentioned problems, and aims to provide a new medical use of paris saponin VII or its salt, and a pharmaceutical composition containing paris saponin VII or paris saponin VII salt.

[0008] The first aspect of the present invention is to provide the use of Paris polyphylla saponin VII or its salt in the preparation of a drug for treating myelodysplastic syndrome. Preferably, the drug for treating myelodysplastic syndrome is a drug that induces ferroptosis of tumor cells.

[0009] More preferably, the drug that induces ferroptosis of tumor cells is a drug that promotes intracellular iron ion accumulation or damages iron homeostasis, causing oxidative stress damage or lipid peroxidation damage to cells.

[0010] The structure of paris saponin VII of the present invention is shown in Formula 1; paris saponin VII salt refers to a pharmaceutically acceptable salt of paris saponin VII, such as hydrochloride, sulfate, citrate, etc.

[0011] The present invention first confirmed through in vitro pharmacodynamic experiments that paridis saponin VII has a significant killing effect on myelodysplastic syndrome tumor cells SKM-1. After intervention with different concentrations of paridis saponin VII for 24h, 48h and 72h, paridis saponin VII significantly inhibited the proliferation of SKM-1 cells (P < 0.01), of which the IC50 at 24h was 0.41μM. At the same time, paridis saponin VII increased ROS levels in a dose-dependent manner (P < 0.01) and could also increase cell Fe 2+level (P<0.01), promoting iron accumulation in tumor cells; after intervention with gradient drug concentrations of Paris polyphylla saponin VII, the level of glutathione (GSH) gradually decreased, and the level of malondialdehyde (MDA) gradually increased (P<0.01, P<0.05), promoting lipid peroxidation and oxidative stress, and dose-dependently down-regulated the protein expression of GPX4 and FTH, impairing intracellular iron homeostasis.

[0012] The use of paris saponin VII or its salt in the preparation of a drug for treating myelodysplastic syndrome of the present invention also has the following technical characteristics: the drug for treating myelodysplastic syndrome is a pharmaceutical composition with paris saponin VII or paris saponin VII salt as the only active ingredient or comprising paris saponin VII or paris saponin VII salt, wherein the content of paris saponin VII or paris saponin VII salt is 0.1-99wt%.

[0013] In addition, the medicine or pharmaceutical composition of the present invention can be prepared into any dosage form with commonly used pharmaceutical excipients, such as common tablets, capsules, granules, injections, decoctions, powders or pills.

[0014] The second aspect of the present invention is to provide a pharmaceutical preparation for treating myelodysplastic syndrome, which comprises paris saponin VII or paris saponin VII salt as an active ingredient and further comprises pharmaceutically acceptable excipients.

[0015] Functions and Effects of the Invention

[0016] In vitro pharmacodynamic experiments have confirmed that Paris saponin VII has a significant killing effect on myelodysplastic syndrome tumor cells, and its mechanism is to promote iron accumulation in tumor cells, promote lipid peroxidation and oxidative stress, thereby inducing ferroptosis of tumor cells. Therefore, the present invention provides a new basis for Paris saponin VII or its salt to treat myelodysplastic syndrome, and also provides a new research idea for the treatment of myelodysplastic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of Paris polyphylla saponin VII on cell proliferation is shown: A, comparison of cell survival rates when Paris polyphylla saponin VII was applied to SKM-1 cells for 24h, 48h, and 72h, respectively; B, comparison of cell survival rates of control group, Paris polyphylla saponin VII group, Z-VAD-FMK (cell apoptosis inhibitor) group, Ferrostatin-1 (ferroptosis inhibitor group), and Paris polyphylla saponin VII combined with Z-VAD or Ferrostatin-1 group, compared with Control, *P<0.05, **P<0.01, compared with PS VII group; #P<0.05, ##P<0.01;

[0018] Figure 2 The effects of different concentrations of Paris polyphylla saponin VII on ROS are shown;

[0019] Figure 3 The effects of different concentrations of Paris polyphylla saponin VII on intracellular iron ions are shown;

[0020] Figure 4 The results show the effects of different concentrations of Paris polyphylla saponin VII on GSH and MDA levels; compared with Control, *P<0.05, **P<0.01.

[0021] Figure 5 The results show the effect of Paris polyphylla saponin VII on ferroptosis pathway proteins; compared with Control, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

[0023] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0024] 1. Materials

[0025] 1. Cells:

[0026] The human MDS cell line SKM-1 was purchased from Wuhan Pronocell Co., Ltd.

[0027] 2. Reagents and instruments:

[0028] Paris polyphylla saponin VII was purchased from MCE, USA; Z-VAD-FMK, Ferrtin-1 and BODIPY C11 probes were all purchased from MCE, USA; CCK8 detection kit and reactive oxygen species detection kit were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; GPX4, FTH and β-actin rabbit monoclonal antibodies and HRP-anti-rabbit-IgG were all purchased from Shanghai Abima Bio-Pharmaceutical Co., Ltd.; NovoCyte flow cytometer was purchased from Agilent, USA; ChemiDoc MP Imaging System multifunctional imaging systems were purchased from Bio-rad, USA; 30086376 Spark microplate reader was purchased from Tecan, Australia.

[0029] 2. Methods

[0030] 1. Cell Culture and Grouping

[0031] The cells were cultured in RPMI-1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) in a cell culture incubator at 37° C. and 5% CO 2 , and cells in the logarithmic growth phase were used for subsequent experiments.

[0032] The present invention takes tumor cells SKM-1 from patients with myelodysplastic syndrome and divides them into a control group, a low, medium and high dose of Paris polyphylla saponin VII group (0.125 μM, 0.25 μM and 0.5 μM), a Z-VAD-FMK (cell apoptosis inhibitor) group, a Ferrostatin-1 (ferroptosis inhibitor group), and a Paris polyphylla saponin VII combined with Z-VAD-FMK or Ferrostatin-1 group.

[0033] CCK-8 was used to detect cell proliferation and evaluate drug toxicity. Spectrophotometry was used to detect glutathione (GSH) and malondialdehyde (MDA) levels, lipid peroxidation levels were evaluated, and flow cytometry was used to detect reactive oxygen species (ROS) and Fe 2+ The intracellular oxidative stress and iron ion levels were evaluated, and the expression levels of GPX4 and FTH proteins involved in the ferroptosis pathway were detected by Western blot.

[0034] 2. CCK-8 test

[0035] Adjust the SKM-1 cell density to 1×10 5 Inoculate in 96-well plates, set as blank group, control group (0μM), Paris polyphylla saponin VII group (0.5μM, 1.0μM, 1.5μM, 2.0μM, 2.5μM), each group set 6 replicates for intervention for 24, 48 and 72h; set blank group, control group (0mg / L), drug intervention group (0.125μM, 0.25μM, 0.5μM), add or not add Z-VAD-FMK (5μM) or Fer-1 (5μM) 2h in advance for pretreatment. Before testing, add 10μl CCK-8 reagent to each well and incubate at 37℃ for 2h, detect the absorbance value (OD) at 450nm with an enzyme reader, and calculate the cell viability (%) according to the formula = [(A (加药) -A (空白) ) / (A (0加药) -A (空白) )]×100%.

[0036] 3. Reactive oxygen species (ROS) activity detection

[0037] After drug intervention in each group, the culture medium was washed away with PBS, and the cells were stained with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min. After the cells were collected and washed, they were centrifuged at 800g for 5 min after staining, and the supernatant was discarded. The cells were then washed twice with PBS, and 200 μL PBS was added to resuspend the cells for flow cytometry analysis.

[0038] 4. Western blot to detect protein levels

[0039] The cells of each group after drug intervention were collected, lysed on ice by adding RIPA, and the protein supernatant was collected by centrifugation for BCA protein quantification. The loading buffer was added and kept in a metal bath at 100°C for 10 minutes. After electrophoresis, transfer, and blocking, GPX4 (1:1000), FTH (1:1000), and β-actin (1:1000) primary antibodies were incubated on a shaker at 4°C overnight. After washing the membrane, HRP-anti-rabbit-IgG (1:10000) secondary antibodies were incubated on a shaker at room temperature for 1 hour. After washing the membrane again, ECL luminescent solution was added and developed with a chemiluminescent imager, and grayscale values ​​were calculated using ImageJ.

[0040] 3. Statistical Analysis

[0041] In this experiment, at least three independent experiments were repeated, and the results were analyzed using GraphPad Prism 10.0 software. The data between two groups were compared using t-test, and the data between multiple groups were compared using one-way analysis of variance. One-way analysis of variance was used for comparison among multiple groups, and P < 0.05 indicated statistically significant differences.

[0042] IV. Results

[0043] 1. Effect of Paris polyphylla saponin VII on cell proliferation

[0044] Different concentrations of Paris polyphylla saponin VII were used to intervene in SKM-1 cells for 24h, 48h and 72h, respectively. The results showed that Paris polyphylla saponin VII could significantly inhibit the proliferation of SKM-1 cells (P<0.01). The IC50 of Paris polyphylla saponin VII on SKM-1 cells at 24h was 0.41μM ( Figure 1 A). To clarify the mode of cell death, the apoptosis inhibitor Z-VAD-FMK was first administered. Z-VAD-FMK could only partially reverse the cell death of SKM-1 cells induced by Paris saponin VII (0.5 μM) (P < 0.05), suggesting that Paris saponin VII has the effect of inducing cell death other than apoptosis. Further pretreatment with the ferroptosis inhibitor Fer-1 significantly reversed the cell death of SKM-1 cells induced by Paris saponin VII (0.5 μM) (P < 0.01), suggesting that Paris saponin VII may induce ferroptosis of SKM-1 cells ( Figure 1 B).

[0045] 2. Effect of Paris polyphylla saponin VII on ROS

[0046] After 24 h of intervention of SKM-1 cells with different concentrations of Paris polyphylla saponin VII, flow cytometry was used to detect ROS levels. The results showed that Paris polyphylla saponin VII induced ROS elevation in a dose-dependent manner. Figure 2 . This suggests that Paris polyphylla saponin VII causes oxidative stress damage to cells.

[0047] 3. Effect of Paris polyphylla saponin VII on intracellular iron ions

[0048] The CalceinAM probe undergoes fluorescence quenching after binding to iron ions. Flow cytometry results show that the fluorescence intensity gradually weakens with increasing drug concentration. Figure 3 This suggests that Paris polyphylla saponin VII causes ferroptosis by promoting the accumulation of intracellular iron ions.

[0049] 4. Effects of Paris polyphylla saponin VII on GSH and MDA

[0050] After intervention with different concentrations of Rhizoma Polygoni Multiflori VII, the antioxidant reducing agent glutathione GSH decreased significantly after 24 hours of high-dose Rhizoma Polygoni Multiflori VII administration (P < 0.01), and the lipid peroxidation product MDA level increased significantly after 24 hours of high-dose Rhizoma Polygoni Multiflori VII administration (P < 0.01). Figure 4 . This suggests that Paris polyphylla saponin VII induces lipid peroxidation damage.

[0051] 5. Effects of Paris polyphylla saponin VII on ferroptosis pathway proteins

[0052] In order to further clarify that Paris polyphylla saponin VII induces ferroptosis in SKM-1 cells, different concentrations of SKM-1 cells were intervened for 24 h. The results showed that the expression of FTH and GPX4 proteins decreased with the increase of drug concentration, and there was a statistically significant difference (P < 0.05, P < 0.01). Figure 5 . This suggests that Paris polyphylla saponin VII impairs iron homeostasis and induces ferroptosis.

[0053] V. Discussion

[0054] Ferroptosis is an iron-dependent cell death mode, which is mainly caused by lipid peroxidation and cell membrane damage. The core mechanism is the irreversible destruction of the cell membrane through the accumulation of ROS and lipid peroxidation. Specifically, the iron-catalyzed Fenton reaction accelerates the production of ROS, and GSH depletion weakens the scavenging ability of GPX4 to lipid peroxides. Lipid peroxidation products (such as MDA) further destroy the cell membrane structure and react with other important macromolecules (such as proteins and DNA) in the cell, leading to cell death. Therefore, the present invention detects the key molecules of the ferroptosis pathway, including iron ions, ROS, GSH, MDA, GPX4 and FTH, to evaluate the tumor cell death mechanism of Paris polyphylla saponin VII in the treatment of myelodysplastic syndrome.

[0055] There are many ways for drugs to induce cell death. This invention is the first to clarify that Paris polyphylla saponin VII can significantly induce the death of myelodysplastic syndrome tumor cells SKM-1. Reactive oxygen species (ROS) are oxygen-containing compounds produced during cell metabolism, including superoxide (O2 - ), hydrogen peroxide (H2O2) and hydroxyl radicals (·OH). In the process of ferroptosis, free iron ions undergo Fenton reaction to release ROS. The generation and accumulation of ROS are important triggers for cells to enter a state of death. The present invention shows that Paris polyphylla saponin VII induces ROS accumulation in a dose-dependent manner, suggesting that cell death is caused by oxidative stress damage. Ferritin is responsible for storing iron in cells and converts iron into Fe 3+ The form is safely stored in its cavity to prevent iron ions from freely participating in the Fenton reaction. The present invention shows that Paris saponin VII downregulates the expression of ferritin FTH, and the level of free iron ions increases, suggesting that Paris saponin VII damages iron homeostasis. Glutathione peroxidase 4 (GPX4) is a key enzyme that inhibits ferroptosis. It can reduce lipid peroxides caused by ROS and prevent cell membranes from being damaged by oxidation. However, the activity of GPX4 depends on GSH as an electron donor. When the level of GSH in the cell decreases, GPX4 is inactivated and cannot effectively remove lipid peroxides, resulting in lipid peroxidation accumulation, which in turn causes ferroptosis. The present invention shows that Paris saponin VII downregulates GPX4 expression and GSH is depleted, suggesting that Paris saponin VII causes lipid peroxidation in SKM-1 cells. MDA is one of the main products of the decomposition of polyunsaturated fatty acids after peroxidation. When lipid peroxidation intensifies during ferroptosis, the amount of MDA generated increases significantly. The present invention shows that Paris saponin VII promotes the accumulation of MDA, suggesting that lipid damage to the cell membrane occurs, marking the occurrence of ferroptosis.

[0056] In summary, the present invention shows that Paris saponin VII (PS VII) has a significant anti-myelodysplastic syndrome tumor effect, and its mechanism of action is to downregulate GPX4 and FTH proteins, impair intracellular iron homeostasis, lead to increased ROS levels, GSH depletion, and increased MDA, which promotes a chain reaction of lipid peroxidation, ultimately destroying the cell membrane and leading to cell ferroptosis.

[0057] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of Paris polyphylla saponin VII or its salt in the preparation of a drug for the treatment of myelodysplastic syndrome.

2. The use according to claim 1, characterized in that: in, The drug for treating myelodysplastic syndrome is a drug that induces ferroptosis of tumor cells.

3. The use according to claim 2, characterized in that: in, The drug that induces ferroptosis of tumor cells is a drug that causes oxidative stress damage or lipid peroxidation damage to cells, promotes intracellular iron ion accumulation, or damages iron homeostasis.

4. The use according to any one of claims 1 to 3, characterized in that: in, The drug for treating myelodysplastic syndrome is a pharmaceutical composition comprising paris saponin VII or paris saponin VII salt.

5. The use according to claim 4, characterized in that: in, The drug for treating myelodysplastic syndrome uses paris saponin VII or paris saponin VII salt as the only active ingredient.

6. The use according to claim 4 or 5, characterized in that: in, The content of paris saponin VII or paris saponin VII salt in the drug for treating myelodysplastic syndrome is 0.1-99wt%.

7. The use according to claim 1, characterized in that: in, The myelodysplastic syndrome therapeutic drug is in the form of tablets, capsules, granules, injections, decoctions, powders or pills.

8. A pharmaceutical composition for treating myelodysplastic syndrome, characterized in that: The invention consists of paris saponin VII or paris saponin VII salt and pharmaceutically acceptable excipients.

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