Application of natural product Withangulin A in preparation of erythroleukemia induced differentiation treatment medicine

Abstract

The invention discloses an application of a natural product WithangatinA in preparation of erythroleukemia induced differentiation treatment drugs, the natural product WithangatinA promotes HEL and K562 cells to be transformed from malignant proliferative cells to normal megakaryocytes by inducing the cell particle size to become larger and significantly increasing multinuclear megakaryocytes and promoting expression of surface labeled molecules differentiated from megakaryocytes at the same time, and the erythroleukemia induced differentiation treatment drugs are obtained. The effect of resisting erythroleukemia is achieved. After the natural product Withangulin A is adopted for treatment, the size and the weight of the spleen of a leukemia mouse can be remarkably reduced, the hematocrit and the platelet number of the mouse are remarkably increased, in addition, treatment of the natural product Withangulin A has no obvious influence on the weight of the mouse, and the safety is high. In-vivo research shows that the medicine can induce megakaryocyte differentiation of erythroleukemia in a mouse body, the effect of resisting erythroleukemia is achieved, and a research basis is provided for development of differentiation treatment medicines of erythroleukemia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a natural product, Withangulatin A, in the preparation of a therapeutic drug for inducing differentiation in erythroleukemia. Background Technology

[0002] Leukemia is a life-threatening malignant tumor caused by the malignant proliferation of white blood cells in the blood or bone marrow, which impairs normal bone marrow hematopoietic function. According to global cancer statistics in 2022, leukemia is the thirteenth most common malignant tumor worldwide and one of the leading causes of cancer-related deaths. Erythroleukemia is a rare and highly aggressive subtype of acute myeloid leukemia, characterized by abnormal malignant proliferation of erythroid precursor cells (such as primitive erythrocytes) in the bone marrow, often accompanied by granulocytic aberrations. Currently, cytarabine combined with anthracyclines is the classic first-line chemotherapy regimen for treating acute myeloid leukemia, but this regimen has drawbacks such as poor selectivity, easy development of drug resistance, and significant side effects. Therefore, further optimization of treatment strategies and development of new treatment methods are needed to improve patients' cure rates and quality of life.

[0003] Differentiation therapy is a novel treatment strategy distinct from traditional chemotherapy. It involves reactivating the cancer cell development process using differentiation-inducing agents, causing tumor cells to differentiate into normal, mature cells, thus losing their malignant phenotype or even completely transforming into normal cells. Differentiation-inducing therapy is an effective method to overcome the shortcomings of radiotherapy and chemotherapy, characterized by its high specificity and minimal side effects. To date, the clinical application of all-trans retinoic acid in the treatment of acute promyelocytic leukemia has demonstrated the effectiveness of differentiation-inducing therapy. However, this therapy is currently only applied clinically to a subtype of acute promyelocytic leukemia, and there are very few drugs available for differentiation-inducing therapy. Therefore, finding and developing other drugs that can induce efficient differentiation of leukemia cells is a pressing need.

[0004] Withangulatin A is a natural product extracted from the nightshade plant *Solanum nigrum*. It belongs to the nightshade lactone class of compounds and has a unique chemical structure and various biological activities. Currently, there are no literature reports on the research and application of Withangulatin A inducing differentiation of erythroleukemia cells and exerting anti-erythroleukemia effects. Summary of the Invention

[0005] The purpose of this invention is to provide a new use for the natural product Withangulatin A.

[0006] This invention provides the application of the natural product Withangulatin A in the preparation of a therapeutic drug for inducing differentiation in erythroleukemia, wherein the natural product Withangulatin A can promote the differentiation of erythroleukemia cells into megakaryocytes.

[0007] The chemical structural formula of the above-mentioned natural product Withangulatin A is shown in Formula I:

[0008]

[0009] The extraction method of the natural product Withangulatin A described in this invention is described in Wang P, Yang XM, Hu ZX, Li YN, Yang J, Hao XJ, Yuan CM, Yi P. UPLC-Q-Orbitrap-MS / MS-Guided Isolation of Bioactive Withanolides from the Fruits of Physalis angulata. J Agric Food Chem. 2023 Nov 8; 71(44):16581-16592. doi:10.1021 / acs.jafc.3c04311.

[0010] The application of the aforementioned natural product Withangulatin A in the preparation of a therapeutic drug for inducing differentiation in erythroleukemia, wherein the erythroleukemia cells include HEL cells and / or K562 cells.

[0011] The application of the aforementioned natural product Withangulatin A in the preparation of a differentiation-inducing therapeutic drug for erythroleukemia, wherein the efficacy of the differentiation-inducing therapeutic drug for erythroleukemia includes at least one of the following:

[0012] (1) In vitro induction of erythroleukemia cells to differentiate into megakaryocytes.

[0013] (2) Inducing erythroleukemia cells to differentiate into megakaryocytes in vivo.

[0014] The aforementioned natural product Withangulatin A induced an increase in the particle size of HEL and K562 cells and a significant increase in multinucleated megakaryocytes. It also promoted the expression of surface markers for megakaryocyte differentiation and facilitated the differentiation of HEL and K562 cells into megakaryocytes.

[0015] The present invention also provides a therapeutic drug for inducing differentiation in erythroleukemia, the drug containing an effective dose of the natural product Withangulatin A, and further comprising pharmaceutically acceptable excipients.

[0016] The dosage form of the drug is any one of tablets, capsules, granules, powders, or liquid preparations.

[0017] The drug can be administered via any one of the following routes: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or oral administration.

[0018] The aforementioned anti-erythroleukemia treatments include acute erythroleukemia.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention provides the application of the natural product Withangulatin A in the preparation of a therapeutic drug for inducing differentiation in erythroleukemia. The natural product Withangulatin A induces cell size increase and significantly increases the number of multinucleated megakaryocytes, while promoting the expression of surface marker molecules for megakaryocyte differentiation, and promoting the transformation of HEL and K562 cells from malignant proliferating cells to normal megakaryocytes, thereby exerting an anti-erythroleukemia effect.

[0021] This invention also provides a differentiation-inducing therapeutic drug for erythroleukemia, the active ingredient of which includes the natural product Withangulatin A, and also includes pharmaceutically acceptable excipients. Treatment with the natural product Withangulatin A significantly reduced the size and weight of the spleen in leukemic mice, and significantly increased the hematocrit and platelet count of the mice. Furthermore, treatment with Withangulatin A had no significant effect on the body weight of the mice, demonstrating high safety. In vivo studies show that the natural product Withangulatin A can induce megakaryocytic differentiation in erythroleukemia in mice, exerting an anti-erythroleukemic effect, providing a research basis for the development of differentiation-inducing therapeutic drugs for erythroleukemia. Attached Figure Description

[0022] Figure 1 These are actual images of HEL cells treated with different concentrations of Withangulatin A.

[0023] Figure 2 These are images of K562 cells treated with different concentrations of Withangulatin A.

[0024] Figure 3 These are Wright-Gymsa staining images of HEL cells treated with different concentrations of Withangulatin A.

[0025] Figure 4 These are Wright-Giemsa staining images of K562 cells treated with different concentrations of Withangulatin A.

[0026] Figure 5 These are staining images of withangulatin A in HEL cells treated with different concentrations of withangulatin A.

[0027] Figure 6 These are staining images of withangulatin A in K562 cells treated with different concentrations of withangulatin A.

[0028] Figure 7 This is a flow cytometry graph showing the detection of megakaryocyte surface marker molecules in HEL cells treated with different concentrations of Withangulatin A. In the graph, A and B represent the effects of different concentrations of Withangulatin A on the proportion of CD41a and CD61 positive cells in HEL cells after treatment for 72 h and 96 h, respectively. C is a statistical graph of the proportion of CD41a positive cells, and D is a statistical graph of the proportion of CD61 positive cells.

[0029] Figure 8 This is a flow cytometry image of megakaryocyte surface marker molecules in K562 cells treated with different concentrations of Withangulatin A. In the image, A and B show the effects of different concentrations of Withangulatin A on the proportions of CD41a and CD61 positive cells in K562 cells after treatment for 72 h and 96 h, respectively. C is a statistical graph showing the proportion of CD41a positive cells, and D is a statistical graph showing the proportion of CD61 positive cells.

[0030] Figure 9 This is a graph showing the effects of Withangulatin A on the baseline indicators of erythroleukemia mice. In the graph, A represents the changes in spleen size in different groups of mice, B represents the changes in spleen weight in different groups, C represents the changes in hematocrit in different groups of mice, and D represents the changes in platelet count in different groups of mice.

[0031] Figure 10 This is a graph showing the effect of Withangulatin A on megakaryocyte surface marker molecules in mouse spleen cells. In the graph, A shows the effect of Withangulatin A on CD41a and CD61, the percentage of CD41a positive cells, and C shows the percentage of CD61 positive cells.

[0032] Figure 11 This is a graph showing the effect of Withangulatin A on the number of megakaryocytes in mouse spleen tissue.

[0033] Figure 12 This is a graph showing the effect of Withangulatin A on the body weight of erythroleukemia mice. Detailed Implementation

[0034] To further illustrate the present invention, the application of the natural product Withangulatin A provided by the present invention in the preparation of a therapeutic drug for inducing differentiation in erythroleukemia is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Cell morphology observation

[0037] HEL cells and K562 cells were respectively divided into 40×10 4 1 piece, 35×10 4 Cells were seeded in 60 mm diameter cell culture dishes and cultured at 37°C for 4 h. After cell stabilization, HEL cells and K562 cells were treated with different concentrations (final concentrations of 0.5, 1, and 1.5 μM) of Withangulatin A, with 0.1% DMSO as the control group. After treatment for 72 h and 96 h, the morphological changes of cells in each group were observed using an inverted microscope. Magnification: 200x, scale bar: 100 μm. Figure 1 and Figure 2 As shown.

[0038] Depend on Figure 1 and Figure 2 It can be seen that with the increase of Withangulatin A concentration and the extension of treatment time, the number of HEL cells and K562 cells decreased, the cell size increased, the irregular shape appeared, and some cells showed adhesion.

[0039] 2. Wright-Gymsa staining

[0040] HEL cells and K562 cells were respectively divided into 40×10 4 1 piece, 35×10 4 HEL cells and K562 cells were seeded in 60 mm diameter cell culture dishes. Different concentrations (final concentrations of 0.5, 1, and 1.5 μM) of Withangulatin A were added to both cells, with 0.1% DMSO as the control. Cells were treated at 37°C for 72 h and 96 h, respectively, and then collected. Cells were transferred to 15 mL centrifuge tubes, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS in 1.5 mL EP tubes. After centrifugation at 1500 rpm for 5 min, 100 μL of PBS was added for resuspending. 10 μL of the cell suspension was scraped onto a clean glass slide and placed horizontally for 30 min to air dry. Wright-Giemsa stain was added to the slide until the cells were completely covered. The slide was allowed to stand for 1.5 min, then an equal volume of PBS was added to dilute the stain. The slide was allowed to stand for 3.5 min, and the stain was gently rinsed away with water. After air drying, the cells were observed under a microscope. The results are shown below. Figure 3 and Figure 4 As shown.

[0041] Wright-Gymsa staining results showed that, compared with DMSO, HEL cells treated with Withangulatin A (…) were significantly less effective. Figure 3 ) and K562 cells ( Figure 4 The cells exhibit increased particle size, multinucleation, and enlarged nuclei, with these changes being time- and concentration-dependent. HEL cells and K562 cells are progenitor cells of the megakaryotic lineage and can differentiate into it. These phenomena are typical of megakaryotic differentiation.

[0042] 3. Rhodamine-labeled phalloidin staining

[0043] HEL cells and K562 cells were divided into 15×10 4 Cells were seeded in 12-well plates, and HEL and K562 cells were treated with different concentrations (final concentrations of 0.5, 1, and 1.5 μM) of Withangulatin A, with 0.1% DMSO as a control. Cells were incubated at 37°C for 72 h, and then collected. Cells were transferred to 15 mL centrifuge tubes, centrifuged at 1500 rpm to remove the supernatant, washed twice with PBS preheated to 37°C, fixed overnight with 4% paraformaldehyde, washed twice with PBS, permeabilized with 0.5% Triton, and then stained with 200 μL of rhodamine-labeled phalloidin for 30 min, followed by counterstaining with DAPI for 5 min. Morphological changes were observed using an inverted fluorescence microscope.

[0044] Phalloidin staining results showed that, compared with DMSO, HEL cells treated with Withangulatin A ( Figure 5 ) and K562 cells ( Figure 6 The cytoplasm of the cells is significantly enlarged, and the nucleus / cytoplasm ratio gradually decreases in a concentration-dependent manner. This phenomenon is also a typical characteristic of megakaryotic differentiation.

[0045] 4. Detection of megakaryocyte surface marker molecules

[0046] HEL cells and K562 cells were respectively divided into 40×10 4 1 piece, 35×10 4HEL cells and K562 cells were seeded in 60 mm diameter cell culture dishes. Different concentrations (final concentrations of 0.5, 1, and 1.5 μM) of Withangulatin A were added to both cells, with 0.1% DMSO as a control. Cells were treated at 37°C for 72 h and 96 h, respectively, and then collected. Cells were transferred to 15 mL centrifuge tubes, centrifuged at 1500 rpm for 5 min, washed once with pre-chilled PBS, and resuspended in 100 μL PBS. 0.5 μL of CD41a-APC and CD61-APC antibodies were added to each tube, mixed well, and incubated at 4°C in the dark for 30 min. The mixture was then filtered through a 300-mesh sieve and analyzed by flow cytometry. Results are shown below. Figure 7 and Figure 8 As shown.

[0047] Depend on Figure 7 It can be seen that after treating HEL cells with Withangulatin A at a final concentration of 1.5 μM for 96 h, compared with the DMSO group, the proportion of CD41a positive cells increased from 43.49% ± 3.83% to 76.08% ± 0.73%, and the proportion of CD61 positive cells increased from 41.14% ± 1.50% to 59.53% ± 0.99%.

[0048] Depend on Figure 8 It can be seen that after treating K562 cells with Withangulatin A at a final concentration of 1.5 μM for 96 h, compared with the DMSO group, the proportion of CD41a positive cells increased from 4.46% ± 0.55% to 22.01% ± 0.33%, and the proportion of CD61 positive cells increased from 4.17% ± 0.34% to 41.51% ± 0.20%.

[0049] The expression levels of megakaryocyte differentiation surface marker molecules were detected by Wright-Gymsa staining, phalloidin staining, and flow cytometry, revealing that Withangulatin A can induce megakaryocyte differentiation in HEL cells and K562 cells.

[0050] 5. Validation of the effect of withangulatin A on megakaryocyte differentiation using a mouse model of erythroleukemia.

[0051] Mouse-derived erythroleukemia cells CB3 were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cells were collected after passage to a stable state and near 80% confluence, and resuspended in physiological saline to achieve a cell density of 80 × 10⁻⁶ cells / mL. 4100 μL of BALB / mL CB3 cell suspension was implanted into 6-week-old BALB / c mice via tail vein. Mice with established models were divided into model groups and treatment groups. The treatment groups were further divided into low-dose groups (1 mg Withangulatin A / kg mice) and high-dose groups (2 mg Withangulatin A / kg mice). A control group (n=6 mice) was also included.

[0052] After modeling was completed, the mice were administered drugs according to their groups the following day via intraperitoneal injection. The dosage of drug solution was based on the mice's body weight. The normal control group received no treatment, the model group received an equal volume of drug solution, the low-dose group received 1 mg Withangulatin A / kg of mice, and the high-dose group received 2 mg Withangulatin A / kg of mice. Drug administration was repeated every other day.

[0053] After 28 days of treatment, the mice were anesthetized and euthanized, and samples were collected for subsequent experiments.

[0054] (1) Measurement of spleen size and weight in mice

[0055] The size of the mouse spleen was measured with a ruler, and its weight was measured with an electronic balance. The results are as follows: Figure 9 As shown in A and B.

[0056] (2) Hematocrit

[0057] Twenty-eight days after treatment, blood was collected from the posterior ocular venous plexus using a heparinized capillary tube, and centrifuged at 3500 rpm for 15 minutes. The percentage of red blood cells in the whole blood within the capillary was measured and calculated; this percentage is the hematocrit. Changes in hematocrit were compared among the groups, and the results are as follows: Figure 9 As shown in C.

[0058] (3) Platelet count determination

[0059] After treatment, an appropriate amount of anticoagulated whole blood was collected using EDTA anticoagulant tubes, and the platelet count of each group of mice was detected using a fully automated five-part differential hematology analyzer. The results are as follows: Figure 9 As shown in D.

[0060] (4) Flow cytometry detection of CD41a and CD61 levels on the surface of megakaryocytes

[0061] Spleen tissues were collected from mice of different groups, ground and filtered, and spleen cells were collected. Cells were transferred to 15 mL centrifuge tubes, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS into 1.5 mL EP tubes, centrifuged at 1500 rpm for 5 min, and then resuspended in 100 μL of PBS. 1 μL of CD41a-FITC and CD61-FITC antibodies were added to each tube, mixed well, and incubated at 4°C in the dark for 30 min. The mixture was then filtered through a 300-mesh sieve, and finally analyzed by flow cytometry. Results are shown below. Figure 10 As shown.

[0062] (5) Effect of hematoxylin-eosin staining on megakaryocyte differentiation in mouse spleen cells

[0063] Spleen tissues from different groups of erythroleukemia mice were collected and fixed in 4% paraformaldehyde. Paraffin sections were obtained through a series of procedures, including dehydration, cleaning, embedding, and sectioning. The sections were then stained with hematoxylin-eosin solution. Finally, the sections were fixed with neutral resin for microscopic observation and imaging. Results are as follows: Figure 11 As shown.

[0064] (6) Mouse weight measurement

[0065] Weighing oneself using an electronic balance, the result is as follows: Figure 12 As shown.

[0066] Each experiment was independently repeated three times. All data were expressed as mean ± SD and analyzed using GraphPad Prism6. Independent samples t-tests were used for comparisons between two samples, and one-way ANOVA was used for comparisons between multiple samples. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001.

[0067] Depend on Figure 9 As shown in Figures A and B, compared to the normal group, the spleen volume and weight in the model group were significantly increased, indicating the successful establishment of the erythroleukemia mouse model. Furthermore, both 1 mg Withangulatin A / kg and 2 mg Withangulatin A / kg doses significantly reduced spleen volume and weight in erythroleukemia mice. Figure 9As shown in Figure C, compared with the normal control group, the hematocrit of the model group was significantly decreased, while the hematocrit of the mice in the Withangulatin A treatment group was significantly increased compared with the model group. Simultaneously, compared with the normal control group, the platelet count of the model group was decreased. Treatment with 1 mg Withangulatin A / kg mice and 2 mg Withangulatin A / kg mice significantly upregulated the platelet count in erythroleukemia mice. Figure 9 (D). These results indicate that Withangulatin A can improve the symptoms of erythroleukemia mice and has a strong anti-erythroleukemia effect in vivo.

[0068] Depend on Figure 10 As can be seen from the flow cytometry results, compared with the normal control group, the proportion of CD41a and CD61 positive cells in the spleen tissue of the model group was significantly reduced. When treated with Withangulatin A, the proportion of CD41a and CD61 positive cells in the spleen tissue of mice was significantly upregulated.

[0069] Depend on Figure 11 It can be seen that HE staining of mouse spleen showed that, compared with the normal control group, the number of megakaryocytes in the spleen tissue of the model group was less. After treatment with Withangulatin A, the number of megakaryocytes in the spleen tissue of mice increased significantly.

[0070] Depend on Figure 12 As can be seen, compared to the model group, the Withangulatin A treatment group had no significant effect on mouse body weight. Body weight is one of the physiological indicators of mice, indirectly reflecting their health status. This indicates that Withangulatin A has no significant toxicity at the effective concentration in erythroleukemia mice.

[0071] Therefore, treatment with Withangulatin A provided by this invention can reduce the volume and weight of the spleen in mice with erythroleukemia, and significantly increase the hematocrit and platelet count. Simultaneously, the proportion of CD41a and CD61 positive cells in the spleen of mice treated with Withangulatin A was significantly upregulated, and the Withangulatin A treatment group had no significant effect on mouse body weight, demonstrating high safety. Based on these results, the natural product Withangulatin A, as a small molecule compound for treating erythroleukemia, can be used to target this specific tumor. It can efficiently induce erythroleukemia cells to differentiate into functional normal benign cells in vivo or in vitro, thereby achieving a therapeutic effect. This provides new ideas and possibilities for the clinical treatment of erythroleukemia, especially acute erythroleukemia, and provides a research foundation for the search and development of therapeutic drugs that induce erythroleukemia differentiation.

Claims

1. The use of a natural product Withangulatin A in the preparation of a drug for inducing differentiation of erytholeukemia, wherein the structure of the natural product Withangulatin A is shown as follows: 2.The use of natural product Withangulatin A in the preparation of a drug for treating red and white blood cell leukemia induction differentiation, characterized in that: The erytholeukemia cells include HEL cells and / or K562 cells. 3.The use of natural product Withangulatin A in the preparation of a drug for treating red and white blood cell leukemia induction differentiation, characterized in that: The natural product Withangulatin A induces differentiation of the erytholeukemia cells into megakaryocytes.

4. A differentiation-inducing therapeutic drug for erythroleukemia, characterized in that: The drug contains an effective dose of the natural product Withangulatin A.

5. The erythroleukemia-inducing differentiation therapeutic drug according to claim 4, characterized by: The drug further includes pharmaceutically acceptable excipients.

6. The erythroleukemia-inducing differentiation therapeutic drug according to claim 4, characterized by: The dosage form of the drug is any one of a tablet, a capsule, a granule, a powder, or a liquid preparation.

7. The erythroleukemia-inducing differentiation therapeutic drug according to claim 4, characterized by: The administration route of the drug is any one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or oral administration.

8. The erythroleukemia-inducing differentiation therapeutic drug according to claim 4, characterized by: The erytholeukemia is acute erytholeukemia.

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