Application of compound Aglaroxin C in preparation of erythroleukemia induced differentiation treatment medicine

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a compound Aglaroxin C in preparation of an erythroleukemia induced differentiation treatment medicine. The compound Aglaroxin C disclosed by the invention has the activity of resisting erythroleukemia cells HEL and K562, and the proliferation of the HEL and K562 cells is inhibited by inducing the HEL and K562 cells to generate G2 phase cycle arrest and cell apoptosis. According to the Aglaroxin C disclosed by the invention, the grain sizes of HEL and K562 cells are induced to become larger, multinuclear cells are remarkably increased, and meanwhile, the expression of a surface marker differentiated from a megakaryocyte line is promoted, so that the HEL and K562 cells are promoted to differentiate to the megakaryocyte line.

Description

Technical Field

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

[0002] Leukemia is a type of malignant tumor that affects human life and health due to the malignant proliferation and differentiation of hematopoietic cells. Leukemia is divided into several types, among which erythroleukemia (erythroleukemia) is a rare subtype of acute myeloid leukemia. Due to the high malignancy and mortality rate of erythroleukemia, there is an urgent need to find new drugs to treat this type of leukemia. Traditional chemotherapy drugs mainly act as cytotoxic agents to induce the death of leukemia cells. Although they have some therapeutic effect, they also cause adverse reactions in normal cells.

[0003] Aglaroxin C is a Roc-A derivative that exhibits strong cytotoxicity against cells infected with hepatitis C virus. Existing technologies disclose the inhibitory effect of Aglaroxin C on the in vitro and in vivo growth of liver cancer cells (see: Xiao Xuezhen, Yuan Dongfang, Xin Dandan, et al. Inhibitory effect of Aglaroxin C on the in vitro and in vivo growth of liver cancer cells [J]. Journal of Xinxiang Medical College, 2018, 35(9): 761-765, 770.). However, the technology regarding whether Aglaroxin C can induce differentiation in erythroleukemia cells has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of compound Aglaroxin C in the preparation of a therapeutic drug for inducing differentiation in erythroleukemia. The compound has anti-erythroleukemia cell activity, can significantly inhibit cell proliferation, promote cell differentiation into megakaryocytes, transform malignant cells into benign cells, and has high safety.

[0005] This invention provides the application of compound Aglaroxin C in the preparation of therapeutic drugs for inducing differentiation in erythroleukemia.

[0006] Preferably, the chemical formula of the compound Aglaroxin C is shown in Formula I:

[0007]

[0008] Preferably, the cells involved in the erythroleukemia include HEL cells and / or K562 cells.

[0009] Preferably, the compound Aglaroxin C has an effect on the IC50 of the HEL cells. 50 The IC50 value for the K562 cells was 29.69 ± 0.55 nM. 50It is 44.26 ± 13.6 nM.

[0010] Preferably, the erythroleukemia differentiation-inducing therapeutic drug includes at least one of the following effects:

[0011] (1) Anti-erythroleukemia cell activity;

[0012] (2) Inhibits the proliferation of erythroleukemia cells;

[0013] (3) Inducing erythroleukemia cells to differentiate into megakaryocytes.

[0014] Preferably, the induction includes upregulating the mRNA levels of megakaryocyte differentiation-related transcription factors in HEL cells and / or K562 cells.

[0015] Preferably, the transcription factors include one or more of JUN, JUNB, JUND, FOS, FOSL1, EGR2, ETS2, GATA2, RUNX1, and TAL1.

[0016] The present invention also provides a therapeutic drug for inducing differentiation in erythroleukemia, wherein the active ingredient of the drug includes the compound Aglaroxin C, and also includes pharmaceutically acceptable excipients.

[0017] Preferably, the anti-erythroleukemia treatment includes anti-acute erythroleukemia treatment.

[0018] Preferably, the dosage of the drug, calculated in mice, is 2–4 mg Aglaroxin C / kg mouse.

[0019] Beneficial Effects: This invention provides the application of compound Aglaroxin C in the preparation of a therapeutic drug for inducing differentiation in erythroleukemia. Aglaroxin C possesses activity against erythroleukemia cells HEL and K562, and inhibits their proliferation by inducing G2 phase cell cycle arrest and apoptosis in HEL and K562 cells. Aglaroxin C of this invention induces increased cell size and a significant increase in multinucleated cells in HEL and K562 cells, while simultaneously promoting the expression of surface markers for megakaryocyte differentiation, thus promoting the differentiation of HEL and K562 cells into normal megakaryocytes and transforming malignant cells into benign cells, thereby exerting an anti-erythroleukemic effect. Treatment with Aglaroxin C of this invention significantly prolongs the survival time of leukemia mice, reduces the size and weight of the spleen, and significantly increases the hematocrit and platelet count in mice. Furthermore, Aglaroxin C treatment has no significant effect on mouse body weight, demonstrating high safety. Further mechanistic studies indicate that Aglaroxin C can upregulate the transcription of megakaryocyte-specific genes through transcription factors including JUN, JUNB, JUND, FOS, FOSL1, EGR2, ETS2, GATA2, RUNX1, and TAL1, thereby inducing HEL and K562 cells to differentiate into megakaryocytes, transforming malignant cells into benign cells, and exerting an anti-erythroleukemia effect.

[0020] This invention also provides a differentiation-inducing therapeutic drug for erythroleukemia. This drug can inhibit the proliferation of HEL and K562 cells in vitro by inducing G2 phase cell cycle arrest and apoptosis, while simultaneously promoting megakaryocytic differentiation, thereby inhibiting the development and progression of erythroleukemia cells. Further in vivo studies show that the drug can induce megakaryocytic differentiation in erythroleukemia cells in vivo, transforming malignant cells into benign cells and exerting an anti-erythroleukemia effect, providing a research basis for the development of differentiation-based therapeutic drugs for erythroleukemia. Attached Figure Description

[0021] Figure 1 Images of HEL cells treated with different concentrations of Aglaroxin C;

[0022] Figure 2 Images of K562 cells treated with different concentrations of Aglaroxin C;

[0023] Figure 3 Cell growth curves of HEL cells treated with different concentrations of Aglaroxin C;

[0024] Figure 4 Cell growth curves of K562 cells treated with different concentrations of Aglaroxin C;

[0025] Figure 5 Cell cycle distribution of HEL cells treated with different concentrations of Aglaroxin C is shown in Figure A, where A represents the effect of different concentrations of Aglaroxin C on the HEL cell cycle after 48 h and 72 h of treatment, B represents the statistical distribution of cell cycle proportions (48 h), and C represents the statistical distribution of cell cycle proportions (72 h).

[0026] Figure 6 Cell cycle distribution diagrams of K562 cells treated with different concentrations of Aglaroxin C are shown. In the diagram, A represents the effect of different concentrations of Aglaroxin C on the cell cycle of K562 cells after treatment for 72 h and 96 h, B represents the statistical distribution of cell cycle proportions (72 h), and C represents the statistical distribution of cell cycle proportions (96 h).

[0027] Figure 7 The graph shows the apoptosis statistics of HEL cells treated with different concentrations of Aglaroxin C. In the graph, A represents the effect of different concentrations of Aglaroxin C on HEL cell apoptosis after 48 h and 72 h of treatment, and B represents the statistical proportion of apoptosis.

[0028] Figure 8 The graph shows the apoptosis statistics of K562 cells treated with different concentrations of Aglaroxin C. In the graph, A represents the effect of different concentrations of Aglaroxin C on the apoptosis of K562 cells after 72 h and 96 h of treatment, and B represents the statistical proportion of apoptosis.

[0029] Figure 9 Wright-Giemsa staining image of HEL cells treated with Aglaroxin C;

[0030] Figure 10 Wright-Giemsa staining image of K562 cells treated with Aglaroxin C;

[0031] Figure 11 Flow cytometry images of megakaryocyte surface marker molecules in HEL cells treated with different concentrations of Aglaroxin C. In the images, A and B represent the effects of different concentrations of Aglaroxin C on the proportion of CD41 and CD61 positive cells in HEL cells after treatment for 48 h and 72 h, respectively. C represents the proportion of CD41+ cells, and D represents the proportion of CD61+ cells.

[0032] Figure 12Flow cytometry images of megakaryocyte surface marker molecules in K562 cells treated with different concentrations of Aglaroxin C. In the images, A and B represent the effects of different concentrations of Aglaroxin C on the proportion of CD41 and CD61 positive cells in K562 cells after treatment for 72 h and 96 h, respectively. C represents the proportion of CD41+ cells, and D represents the proportion of CD61+ cells.

[0033] Figure 13 The image shows the qRT-PCR results of Aglaroxin C on megakaryocyte differentiation-related transcription factors in HEL cells.

[0034] Figure 14 The image shows the qRT-PCR results of Aglaroxin C on megakaryocyte differentiation-related transcription factors in K562 cells.

[0035] Figure 15 Survival curves of leukemia mice treated with Aglaroxin C;

[0036] Figure 16 The graph shows the effects of Aglaroxin C on the baseline indicators of leukemia 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 of mice, C represents the changes in hematocrit in different groups of mice, and D represents the changes in platelet count in different groups of mice.

[0037] Figure 17 The figure shows the effect of Aglaroxin C on megakaryocyte surface marker molecules in mouse spleen cells. In the figure, A represents the effect of Aglaroxin C on CD41 and CD61, megakaryocyte surface marker molecules in mouse spleen cells, B represents CD41+ statistics, and C represents CD61+ statistics.

[0038] Figure 18 The figure shows the effect of Aglaroxin C on the body weight of leukemia mice. Detailed Implementation

[0039] This invention provides the application of compound Aglaroxin C in the preparation of therapeutic drugs for inducing differentiation in erythroleukemia.

[0040] In this invention, the chemical formula of the compound Aglaroxin C is shown in Formula I:

[0041]

[0042] The synthesis method of the compound Aglaroxin C described in this invention can be found in Stone, Steven D., et al. "Biomimetic Kinetic Resolution: Highly Enantio- and Diastereoselective Transfer Hydrogenation of Aglain Ketones To Access Flavagline Natural Products." Journal of the American Chemical Society 137.1 (2015): 525-530.

[0043] The cells used to treat erythroleukemia according to the present invention preferably include HEL cells and / or K562 cells, and more preferably HEL cells and K562 cells.

[0044] The compound Aglaroxin C of this invention has an effect on the IC50 of the HEL cells. 50 The preferred concentration is 29.69 ± 0.55 nM, which is the IC50 value for the K562 cells. 50 The preferred concentration is 44.26 ± 13.6 nM. The compound Aglaroxin C described in this invention exhibits activity against HEL and K562 cells in erythroleukemia.

[0045] This invention provides the application of compound Aglaroxin C in the preparation of therapeutic drugs for inducing differentiation in erythroleukemia.

[0046] The erythroleukemia differentiation-inducing therapeutic drug of the present invention has at least one of the following effects:

[0047] (1) Anti-erythroleukemia cell activity;

[0048] (2) Inhibits the proliferation of erythroleukemia cells;

[0049] (3) Inducing erythroleukemia cells to differentiate into megakaryocytes.

[0050] The compound Aglaroxin C described in this invention inhibits the proliferation of HEL and K562 cells by inducing G2 phase cell cycle arrest and apoptosis. Aglaroxin C also induces increased cell size and a significant increase in multinucleated cells in HEL and K562 cells, while simultaneously promoting the expression of surface markers for megakaryocyte differentiation, thus promoting the differentiation of HEL and K562 cells into megakaryocytes.

[0051] The induction method described in this invention preferably includes upregulating the mRNA levels of megakaryocytic differentiation-related transcription factors in HEL cells and / or K562 cells. The transcription factors described in this invention preferably include one or more of JUN, JUNB, JUND, FOS, FOSL1, EGR2, ETS2, GATA2, RUNX1, and TAL1, thereby promoting the differentiation of HEL and K562 cells into megakaryocytic lineages.

[0052] This invention also provides a therapeutic drug for inducing differentiation in erythroleukemia, the active ingredient of which includes the compound Aglaroxin C, and further includes pharmaceutically acceptable excipients. Aglaroxin C, as a small molecule compound for treating erythroleukemia, can be used to target leukemia, a specific type of tumor, by inducing leukemia cells to differentiate into normal cells, thereby achieving a therapeutic effect. Aglaroxin C has no significant effect on mouse body weight and exhibits high safety.

[0053] The anti-erythroleukemia drugs described in this invention preferably include those for acute erythroleukemia. The Aglaroxin C described in this invention can be used in the preparation of drugs to induce differentiation in acute erythroleukemia, providing a new approach for the development of drugs to treat erythroleukemia.

[0054] The dosage of the drug described in this invention is based on mice, preferably 2-4 mg Aglaroxin C / kg mice, more preferably 4 mg Aglaroxin C / kg mice.

[0055] To further illustrate the present invention, the application of the compound Aglaroxin C 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.

[0056] Example 1

[0057] The synthesis method of the compound Aglaroxin C described in this invention can be found in Stone, Steven D., et al. "Biomimetic Kinetic Resolution: Highly Enantio- and Diastereoselective Transfer Hydrogenation of Aglain Ketones To Access Flavagline Natural Products." Journal of the American Chemical Society 137.1 (2015): 525-530.

[0058] Example 2 Cell Viability Test

[0059] HEL and K562 cells were cultured in RPMI-1640 and DMEM media, respectively. A mixture of 10% fetal bovine serum and 1% penicillin / streptomycin was added to the cell culture medium, and the cells were passaged in a 37°C, saturated humidity, and 5% CO2 incubator. Once the cells reached the logarithmic growth phase, HEL and K562 cells were collected and seeded into 96-well plates (HEL cells: 10,000 cells / well; K562 cells: 8,000 cells / well). 90 μL of sterile water was added around each well, and the plates were incubated at 37°C for 4 hours. After the cells stabilized, they were divided into groups of five replicates each. Four groups were selected as treatment groups, each treated with different concentrations (100, 50, 25, and 12.5 nM) of Aglaroxin C. Another group was selected as the control group, treated with 0.1% DMSO. After 72 hours of treatment (at 37°C), 10 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C for 4 hours. Once formazan had fully formed, 100 μL of a triplet solution was added to each well (preparation of the triplet solution: add 100 g SDS, 50 ml isobutanol, and 1 ml concentrated hydrochloric acid to a clean glass beaker, add purified water to 1 L, stir until completely dissolved, and then aliquot and store). The cells were incubated overnight at 37°C. After the formazan had completely dissolved, the absorbance (OD value) at 570 nm was measured using a microplate reader, and the inhibition rate was calculated using the following formula.

[0060] Inhibition rate = (OD value of control group 570nm - OD value of treatment group 570nm) / OD value of control group 570nm.

[0061] The control group received 0.1% DMSO, while the treatment groups received different concentrations of Aglaroxin C (100, 50, 25, and 12.5 nM).

[0062] Half-maximal inhibition rate (IC50) 50 The calculation method for IC50 is as follows: Based on the inhibition rate, the forecast function is used to obtain the IC50 corresponding to the compound. 50 .

[0063] The results showed that different concentrations of Aglaroxin C inhibited the cell viability of HEL and K562 cells in a concentration-dependent manner, and had an IC50 effect on HEL. 50 The value is 29.69 ± 0.55 nM for IC K562. 50 It is 44.26 ± 13.6 nM.

[0064] Example 3: Cell Morphology Observation

[0065] HEL and K562 cells in logarithmic growth phase were collected according to Example 1 and seeded in 96-well plates (HEL cells: 10,000 cells / well; K562 cells: 8,000 cells / well) and cultured at 37°C for 4 hours. After cell stabilization, HEL cells were treated with different concentrations (5, 10, and 20 nM) of Aglaroxin C, with 0.1% DMSO as the control group, for 48 and 72 hours, respectively. K562 cells were treated with different concentrations (10, 20, and 40 nM) of Aglaroxin C, with 0.1% DMSO as the control group, for 72 and 96 hours, respectively. 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.

[0066] Depend on Figure 1 and Figure 2 It can be seen that with the increase of Aglaroxin C concentration and the extension of treatment time, the number of HEL and K562 cells decreased, the cell size increased, and irregular morphology appeared. Furthermore, it can induce the transformation of suspended cells into adherent morphology.

[0067] Example 4: Cell Growth Curve Determination

[0068] HEL and K562 cells in logarithmic growth phase were collected as described in Example 1 and seeded in 96-well plates (HEL cells: 10,000 cells / well; K562 cells: 8,000 cells / well). 90 μL of sterile water was added around each well, and the plates were incubated for 4 hours. After cell stabilization, HEL cells were treated with different concentrations (10, 20, and 40 nM) of Aglaroxin C, and OD values ​​(570 nm) were measured at 0 h, 24 h, 48 h, 72 h, and 96 h. K562 cells were treated with different concentrations (20, 40, and 80 nM) of Aglaroxin C, with 0.1% DMSO as a control. OD values ​​(570 nm) were measured at 0 h, 24 h, 48 h, 72 h, and 96 h. Cell growth curves were plotted. Figure 3 and Figure 4 As shown.

[0069] Depend on Figure 3 and 4 It can be seen that, compared with the DMSO group, different concentrations of Aglaroxin C can inhibit the proliferation of both cell lines, and the growth rate of both cell lines gradually slows down with increasing concentration, indicating that Aglaroxin C can inhibit HEL in a concentration- and time-dependent manner. Figure 3 ) and K562 ( Figure 4 ) proliferation.

[0070] Example 5 Cell cycle detection

[0071] HEL and K562 cells were divided into 5×10 5 Cells were seeded per well in 60 mm diameter cell culture dishes. HEL cells were treated with different concentrations (5, 10, and 20 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 48 h and 72 h, respectively. K562 cells were treated with different concentrations (10, 20, and 40 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 72 h and 96 h, respectively. 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, the supernatant was discarded, and the cells were resuspended in pre-chilled 70% ethanol and fixed overnight at -20°C. Centrifuge at 1300 rpm for 10 min, discard the supernatant, resuspend and wash the cells with PBS, centrifuge again at 1300 rpm for 5 min, discard the supernatant, and resuspend the cells in 500 μL PBS. Add 5 μL RNase A, 25 μL propidium iodide, and 25 μL Triton X-1000 to each sample, mix well, and incubate in a 37°C water bath in the dark for 30 min. Centrifuge at 1000 rpm for 5 min, discard the dye, resuspend in 200 μL PBS, filter through a 300-mesh sieve, and finally analyze using flow cytometry. The results are as follows: Figure 5 and Figure 6 As shown.

[0072] Depend on Figure 5 It can be seen that after HEL cells were treated with high concentrations of Aglaroxin C for 72 hours, the proportion of G2 / M phase cells increased from 17.01% ± 0.26% to 33.69% ± 0.23% compared with the DMSO group.

[0073] Depend on Figure 6 It can be seen that after K562 cells were treated with high concentrations of Aglaroxin C for 96 hours, the proportion of cells in the G2 / M phase increased from 18.25% ± 0.42% to 44.80% ± 0.58% compared with the DMSO group.

[0074] The results showed that the proportion of HEL and K562 cells in the G2 / M phase increased significantly after Aglaroxin C treatment, and this increase was concentration-dependent, indicating that Aglaroxin C can induce G2 / M phase cell cycle arrest in HEL and K562 cells.

[0075] Example 6 Apoptosis Detection

[0076] HEL and K562 cells were divided into 5×105 Cells were seeded per well in 60 mm diameter cell culture dishes. HEL cells were treated with different concentrations (5, 10, and 20 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 48 h and 72 h, respectively. K562 cells were treated with different concentrations (10, 20, and 40 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 72 h and 96 h, respectively. 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, centrifuged at 1500 rpm for 5 min, and then resuspended in 100 μL of 1× binding buffer. Add 5 μL of FITC-Annexin V and 5 μL of PI dye to each sample, mix well, incubate at room temperature in the dark for 15 min, centrifuge at 1000 rpm for 5 min, discard the dye, resuspend in 100 μL of 1× binding buffer, and finally analyze using flow cytometry. The results are as follows: Figure 7 and Figure 8 As shown.

[0077] from Figure 7 It can be seen that after Aglaroxin C was applied to HEL cells, it could induce apoptosis compared with the control group DMSO. The apoptosis rate was highest in the high concentration group of Aglaroxin C (20 nM) at 72 h, and the total apoptosis rate of the high concentration group of Aglaroxin C increased from 10.22% ± 0.58% to 34.01% ± 0.97%.

[0078] from Figure 8 It can be seen that after Aglaroxin C was applied to K562 cells, it could induce apoptosis compared with the control group DMSO. At 96 h, the total apoptosis rate of the high concentration group of Aglaroxin C increased from 6.47% ± 0.69% to 94.02% ± 0.13%, and the apoptosis rate at 96 h was higher than that at 72 h.

[0079] These results indicate that Aglaroxin C induces apoptosis in HEL and K562 cells in a concentration- and time-dependent manner.

[0080] Example 7 Wright-Gymsa staining

[0081] HEL and K562 cells were divided into 5×10 5Cells were seeded per well in 60 mm diameter cell culture dishes. HEL cells were treated with different concentrations (5, 10, and 20 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 48 h and 72 h, respectively. K562 cells were treated with different concentrations (10, 20, and 40 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 72 h and 96 h, respectively. 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, centrifuged at 1500 rpm for 5 min, and then resuspended in 100 μL of PBS. The resuspended cells were scraped onto a clean glass slide, placed horizontally for 30 min, and then air-dried. Add Wright-Gymsa stain to a glass slide until it completely covers the cells. Let it stand for 1 minute, then add an equal volume of PBS to dilute the stain. Let it stand for 5 minutes, then gently rinse away the stain with water. After drying, observe the results under a microscope. Figure 9 and Figure 10 As shown.

[0082] Depend on Figure 9 and Figure 10 As can be seen, compared with DMSO, HEL treated with Aglaroxin C showed... Figure 9 ) and K562 ( Figure 10 The cell size increases, and the percentage of multinucleated cells increases significantly, exhibiting a time- and concentration-dependent effect. HEL and K562 cells are progenitor cells of the megakaryotic lineage and can differentiate into megakaryotic lines. These phenomena are typical of megakaryotic differentiation.

[0083] Example 8: Detection of Marker Molecules on the Surface of Megakaryotic Cells

[0084] HEL and K562 cells were divided into 5×10 5Cells were seeded per well in 60 mm diameter cell culture dishes. HEL cells were treated with different concentrations (5, 10, and 20 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 48 h and 72 h, respectively. K562 cells were treated with different concentrations (10, 20, and 40 nM) of Aglaroxin C, with 0.1% DMSO as a control. Cells were collected after incubation at 37°C for 72 h and 96 h, respectively. 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. 0.5 μL of CD41-FITC and CD61-APC antibodies were added to each tube, mixed well, and incubated at 4°C in the dark for 40 min. The mixture was then filtered through a 300-mesh sieve and analyzed by flow cytometry. Results are shown below. Figure 11 and Figure 12 As shown.

[0085] Depend on Figure 11 It can be seen that after HEL cells were treated with a high concentration (20 nM) of Aglaroxin C for 72 h, compared with the DMSO group, the proportion of CD41+ cells increased from 44.55% ± 0.40% to 94.21% ± 0.74%, and the proportion of CD61+ cells increased from 57.61% ± 0.69% to 86.60% ± 0.55%.

[0086] Depend on Figure 12 It can be seen that after K562 cells were treated with high concentrations of Aglaroxin C for 96 hours, compared with the DMSO group, the proportion of CD41+ cells increased from 6.18%±0.43% to 70.52%±0.30%, and the proportion of CD61+ cells increased from 8.52%±0.75% to 60.00%±0.48%.

[0087] The expression levels of megakaryocyte differentiation surface marker molecules were detected by Wright-Gymsa staining and flow cytometry, revealing that Aglaroxin C can induce megakaryocyte differentiation in HEL and K562 cells.

[0088] Example 9: Detection of mRNA levels of megakaryocyte differentiation-related transcription factors after Aglaroxin C treatment using real-time quantitative PCR (qRT-PCR).

[0089] (1) Extraction of total RNA

[0090] HEL and K562 cells were divided into 5×10 5HEL cells were seeded per well in 60 mm diameter cell culture dishes. HEL cells were treated with a high concentration (20 nM) of Aglaroxin C, and K562 cells were treated with a high concentration (40 nM) of Aglaroxin C for 48 h. Cells were then collected. Total RNA was extracted from the cells according to the TRIzol kit instructions. An appropriate amount of the dissolved total RNA was taken, and its quality was assessed by electrophoresis. RNA concentration was determined using a NanoDrop 2000 micro-spectrophotometer. The cells were stored at -80℃ for later use.

[0091] (2) cDNA was synthesized by reverse transcription using the TaKaRa reverse transcription kit.

[0092] (3) According to the qRT-PCR kit instructions, prepare the reaction system and perform qRT-PCR. The genes detected are differentiation-related genes JUN, JUNB, JUND, FOS, FOSL1, EGR2, ETS2, GATA2, RUNX1, and TAL1. Obtain the relative mRNA level data. The results are as follows: Figure 13 and Figure 14 As shown.

[0093] Depend on Figure 13 and Figure 14 It can be seen that after Aglaroxin C was applied to HEL and K562 cells, the transcription factors related to megakaryocyte differentiation, namely JUN, JUNB, JUND, FOS, FOSL1, EGR2, ETS2, GATA2, RUNX1 and TAL1, showed a significant upregulation trend.

[0094] Example 10: Establishment of a mouse leukemia model, Aglaroxin C treatment, and data collection.

[0095] 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 8 × 10⁻⁶ cells / mL. 5 100 μL of CB3 cell suspension was implanted into 6-week-old BALB / c mice via tail vein injection. Mice with established models were divided into model groups and treatment groups. The treatment groups included a low-dose group (2 mg Aglaroxin C / kg mice) and a high-dose group (4 mg Aglaroxin C / kg mice). A control group (no model group) was also included. Each control group was further randomly divided into two subgroups: one for tissue sampling (n=6 per group) and the other for observing mouse survival (n=10 per group).

[0096] After modeling was completed, the mice were administered drugs according to their groups the following day via intraperitoneal injection. Each mouse received 200 μL of the drug solution. The control group received no treatment, the model group received an equal volume of drug solution, the low-dose group received 2 mg Aglaroxin C / kg, and the high-dose group received 4 mg Aglaroxin C / kg. Drug administration was performed every other day. Mice survival was observed and recorded daily, and survival curves were plotted. The results are shown below. Figure 15 As shown.

[0097] After 21 days of treatment, the mice were anesthetized and euthanized for sampling and subsequent experiments.

[0098] ① Measurement of spleen size and weight in mice

[0099] 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 16 As shown in A and B.

[0100] ② Hematocrit assay

[0101] Twenty-one 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 volume 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 16 As shown in C.

[0102] ③ Platelet count determination

[0103] 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 16 As shown in D.

[0104] ④ Flow cytometry detection of CD41 and CD61 levels on the surface of megakaryocytes

[0105] 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 in 1.5 mL EP tubes. After centrifugation at 1500 rpm for 5 min, 100 μL of PBS was added for resuspending. 0.5 μL of CD41-FITC and CD61-FITC antibodies were added to each tube, mixed well, and incubated at 4°C in the dark for 40 min. The mixture was then filtered through a 300-mesh sieve, and finally analyzed by flow cytometry. Results are shown below. Figure 17 As shown.

[0106] ⑤ Mouse weight measurement

[0107] Weighing oneself using an electronic balance, the result is as follows: Figure 18 As shown.

[0108] Each experiment was independently repeated three times. All data were expressed as mean ± SD and analyzed using GraphPad Prism 6. 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.

[0109] Depend on Figure 15 As can be seen from the daily observation and recording of mouse survival, and the plotting of survival curves, all mice in the model group died on day 47. After day 108, the survival rate of the low-dose group was 20%, and after day 80, the survival rate of the high-dose group was 50%. Compared with the model group, high-dose Aglaroxin C significantly prolonged the survival time of leukemia mice (P = 0.0176).

[0110] Depend on Figure 16 As shown in Figures A and B, the spleen size and weight in the mice are significantly larger in the model group compared to the normal group, indicating that the leukemia mouse model has been successfully established. Furthermore, doses of 2 mg Aglaroxin C / kg and 4 mg / kg Aglaroxin C / kg in mice significantly reduced spleen volume and weight in leukemia mice. Figure 16 As can be seen from the results, compared with the normal control group, the hematocrit of the model group was significantly reduced, while the hematocrit of the mice in the Aglaroxin C 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 reduced. Treatment with 4 mg Aglaroxin C / kg mice and 2 mg Aglaroxin C / kg mice significantly upregulated the platelet count in leukemia mice. Figure 16 (D). These results indicate that Aglaroxin C can improve the symptoms of leukemia in mice and has a strong anti-leukemia effect in vivo.

[0111] Depend on Figure 17 As can be seen from the flow cytometry results, compared with the normal control group, the proportion of CD41+ and CD61+ cells in the spleen tissue of the model group was significantly reduced. When treated with Aglaroxin C, the proportion of CD41+ and CD61+ cells in the spleen tissue of mice was significantly upregulated.

[0112] Depend on Figure 18 As can be seen, compared to the model group, the Aglaroxin C treatment group had no significant effect on the mouse body weight. Body weight is one of the physiological indicators of mice, indirectly reflecting their health status.

[0113] Therefore, treatment with Aglaroxin C provided by this invention significantly prolongs the survival time of leukemia mice, reduces the size and weight of the spleen, and significantly increases hematocrit and platelet count. Furthermore, Aglaroxin C treatment significantly upregulates the proportion of CD41+ and CD61+ cells in the spleen of mice, and the treatment group has no significant effect on mouse body weight, demonstrating high safety. Based on these results, Aglaroxin C can serve as a potential small molecule for treating erythroleukemia, providing a research foundation for the development of differentiation therapy drugs for erythroleukemia.

[0114] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of compound Aglaroxin C in the preparation of a differentiation-inducing therapeutic drug for erythroleukemia.

2. The application according to claim 1, characterized in that, The chemical formula of the compound Aglaroxin C is shown in Formula I:

3. The application according to claim 1, characterized in that, The cells involved in the erythroleukemia include HEL cells and / or K562 cells.

4. The application according to claim 3, characterized in that, The compound Aglaroxin C has an effect on the IC50 of the HEL cells. 50 The IC50 value for the K562 cells was 29.69 ± 0.55 nM. 50 It is 44.26 ± 13.6 nM.

5. The application according to claim 1, characterized in that, The erythroleukemia differentiation-inducing therapy drug has at least one of the following effects: (1) Anti-erythroleukemia cell activity; (2) Inhibits the proliferation of erythroleukemia cells; (3) Inducing erythroleukemia cells to differentiate into megakaryocytes.

6. The application according to claim 5, characterized in that, The induction includes upregulating the mRNA levels of megakaryocyte differentiation-related transcription factors in HEL cells and / or K562 cells.

7. The application according to claim 6, characterized in that, The transcription factors include one or more of JUN, JUNB, JUND, FOS, FOSL1, EGR2, ETS2, GATA2, RUNX1, and TAL1.

8. A differentiation-inducing therapeutic drug for erythroleukemia, characterized in that, The active ingredient of the drug includes the compound Aglaroxin C, and also includes pharmaceutically acceptable excipients.

9. The medicament according to claim 8, characterized in that, The anti-erythroleukemia treatment includes treatment for acute erythroleukemia.

10. The medicament according to claim 8, characterized in that, The dosage of the drug, calculated in mice, is 2–4 mg Aglaroxin C / kg mouse.

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