Use of alnusenone or a derivative thereof for the preparation of a medicament for the treatment of thrombocytopenia

Abstract

This invention provides the use of alder ketone or its derivatives in the preparation of drugs for treating thrombocytopenia. Alder ketone does not affect the cell viability of megakaryocytes in vitro, making it safer to use. It also exhibits some activity in vitro, promoting the differentiation and maturation of megakaryocytes and the formation of proplatelet cells. Furthermore, studies have found that alder ketone promotes platelet production in vivo, restoring platelet levels in patients with thrombocytopenia and promoting the differentiation, maturation, and polyploid formation of megakaryocytes in the main hematopoietic tissues of bone marrow, spleen, and lung. Alder ketone has a certain effect in treating thrombocytopenia, and it is widely found in natural Chinese medicinal materials, making it relatively inexpensive and with few side effects.

Description

Technical Field

[0001] This invention relates to the use of alder ketone or its derivatives in the preparation of medicaments for treating thrombocytopenia, and belongs to the pharmaceutical field. Background Technology

[0002] Thrombocytopenia is a common blood disorder. Many factors can cause thrombocytopenia, such as leukemia, aplastic anemia, and spontaneous thrombocytopenia due to immune factors. Furthermore, cancer patients often undergo radiotherapy and chemotherapy, which can eventually lead to thrombocytopenia, forcing many patients to discontinue these treatments. Currently, different treatment principles exist for different causes of thrombocytopenia. For example, drug therapy, including glucocorticoids and immunoglobulins, can be used to treat primary immune thrombocytopenia. Platelet transfusions and dietary therapy can also be used to improve thrombocytopenia caused by platelet dysfunction. However, platelet transfusions can cause a series of adverse reactions, such as decreased body mass index and compensatory splenomegaly. Dietary therapy can improve thrombocytopenia. However, these common therapies are not as effective as drug therapy, and drug therapy is relatively expensive. Currently, there is no definitive treatment for thrombocytopenia caused by radiotherapy and chemotherapy.

[0003] Alnustone (ALN) is found in many natural medicinal materials, such as alder, cardamom, and turmeric. Its main effects are anti-inflammatory and antiemetic, and it is used to treat psoriasis and other skin conditions. Alnustone belongs to the diarylheptane class of compounds, which have varying effects. CN202310117932.3, entitled "Use of Alnustone in Inhibiting and Treating Gastric Cancer," discloses the use of alnustone in inhibiting the proliferation of undifferentiated gastric cancer cells HGC-27. CN202111462413.8, entitled "Application of Alnustone in the Preparation of Drugs for the Prevention and Treatment of Glucose Metabolism Disorders," discloses the application of alnustone in the preparation of drugs and health products for the prevention and treatment of glucose metabolism disorders; the alnustone can be obtained through chemical synthesis or biological extraction; the diseases mentioned include, but are not limited to, type 2 diabetes, gestational diabetes, polycystic ovary syndrome, obesity, and metabolic syndrome. CN202210581569.6, Invention Title: Application of Alderne in the Preparation of Drugs for the Prevention or Treatment of Atopic Dermatitis, discloses the application of alderne in the preparation of drugs for atopic dermatitis, as well as the combined use of alderne with other drugs for atopic dermatitis.

[0004] To date, there is no literature reporting on the therapeutic effect of alder ketone on thrombocytopenia induced by radiotherapy and chemotherapy, nor on its mechanism of action. Summary of the Invention

[0005] This invention relates to novel uses of alder ketone or its derivatives. Specifically, it relates to its use in the preparation of medicaments for treating thrombocytopenia.

[0006] This invention provides the use of alder ketone or its derivatives in the preparation of medicaments for treating thrombocytopenia.

[0007] The structural formula of alderne is:

[0008] The drug mentioned is a medication for treating thrombocytopenia caused by aplastic anemia.

[0009] The aforementioned drug is a drug that restores platelet levels and promotes platelet production in patients with thrombocytopenia.

[0010] The drug mentioned above is a drug that promotes the differentiation and maturation of megakaryocytes in the bone marrow, spleen, and lungs of hematopoietic tissue.

[0011] The present invention also provides a pharmaceutical composition for treating thrombocytopenia, comprising an effective amount of alder ketone or a derivative thereof.

[0012] The pharmaceutical composition of the present invention is a pharmaceutical preparation made by using an effective amount of alder ketone as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients.

[0013] The pharmaceutical preparations mentioned herein are oral preparations, injectable preparations, topical preparations, sustained-release preparations, or controlled-release preparations.

[0014] The oral preparations include capsules, granules, tablets, mixtures, or syrups.

[0015] Among them, pharmaceutically acceptable excipients are saccharide compounds or at least one of fillers, binders, disintegrants, and glidants.

[0016] Furthermore, the carbohydrate compound is at least one selected from lactose, mannitol, starch, glucose, and sorbitol. The filler is at least one selected from lactose, mannitol, microcrystalline cellulose, and starch. The binder is at least one selected from povidone, hydroxypropyl methylcellulose starch, and low-substituted hydroxypropyl cellulose. The disintegrant is at least one selected from croscarmellose sodium, croscarmellose sodium, and croscarmellose. The flow aid is at least one selected from magnesium stearate, talc, silica, and polyethylene glycol 6000.

[0017] Through extensive experimental research by the inventors, alder ketone has been shown to have no effect on the cell viability of megakaryocytes in vitro, making it safer to use. In vitro, it exhibits certain activity in promoting the differentiation and maturation of megakaryocytes and the production of proplatelet cells. Simultaneously, studies have found that alder ketone promotes platelet production in vivo, restoring platelet levels in patients with thrombocytopenia and promoting the differentiation, maturation, and polyploidization of megakaryocytes in the main hematopoietic tissues of bone marrow, spleen, and lungs. Therefore, alder ketone has a certain effect in treating thrombocytopenia, and it is widely found in natural Chinese medicinal materials, making it relatively inexpensive and with few side effects. Attached Figure Description

[0018] Figure 1 The effect of alder ketone on platelet levels in irradiated thrombocytopenic mice (*Compared with the model group, *P<.05, **P<.01, ***P<.001).

[0019] Figure 2 The effect of alder ketone on the mean platelet volume level in irradiated thrombocytopenic mice (*Compared with the model group, *P<.05,**P<.01,***P<.001).

[0020] Figure 3 The figure shows the effect of alder ketone on CD41 / CD61 expression in bone marrow cells of irradiated thrombocytopenic mice.

[0021] Figure 4 The figure shows the statistical analysis of the effect of alder ketone on CD41 / CD61 expression in bone marrow cells of irradiated thrombocytopenic mice (*Compared with the model group, *P<.05, **P<.01, ***P<.001).

[0022] Figure 5 The figure shows the effect of alder ketone on CD41 / CD61 expression in spleen cells of irradiated thrombocytopenic mice.

[0023] Figure 6 The figure shows the statistical analysis of the effect of alder ketone on CD41 / CD61 expression in spleen cells of irradiated thrombocytopenic mice (*Compared with the model group, *P<.05, **P<.01, ***P<.001).

[0024] Figure 7 The figure shows the effect of alder ketone on CD41 / CD61 expression in lung cells of irradiated thrombocytopenic mice. Figure 8 The figure shows the statistical analysis of the effect of alder ketone on CD41 / CD61 expression in lung cells of irradiated thrombocytopenic mice (*Compared with the model group, *P<.05, **P<.01, ***P<.001).

[0025] Figure 9The image shows the H&E staining results of alder ketone on bone marrow cells of mice with irradiated thrombocytopenia.

[0026] Figure 10 The statistical analysis of H&E staining of bone marrow cells from irradiated thrombocytopenic mice by alder ketone is shown in the figure (*Compared with the model group, *P<.05, **P<.01, ***P<.001).

[0027] Figure 11 The image shows the H&E staining results of alder ketone on spleen cells of mice with irradiated thrombocytopenia.

[0028] Figure 12 The statistical analysis of H&E staining of spleen cells from mice with irradiated thrombocytopenia by alder ketone is shown in the figure (*Compared with the model group, *P<.05, **P<.01, ***P<.001).

[0029] Figure 13 Figure showing the effect of alder ketone on platelets in transgenic zebrafish.

[0030] Figure 14 The statistical analysis of alder ketone on platelets in transgenic zebrafish is shown in the figure (*Compared with the control group, *P<.05, **P<.01, ***P<.001).

[0031] Figure 15 Microscopic images showing the effect of alder ketone on the differentiation of Meg-01 and HEL cells.

[0032] Figure 16 Giemsa staining of Meg-01 and HEL cells after alderne treatment.

[0033] Figure 17 Image showing phalloidin staining in Meg-01 cells after alder ketone intervention.

[0034] Figure 18 Image showing phalloidin staining in HEL cells after alderne intervention.

[0035] Figure 19 The figure shows the effect of alder ketone on CD41 / CD42b expression in Meg-01 cells.

[0036] Figure 20 Statistical analysis of alfalfa ketone intervention on CD41 / CD42b expression in Meg-01 cells (*Compared with the control group, *P<.05, **P<.01, ***P<.001).

[0037] Figure 21 The figure shows the effect of alder ketone on CD41 / CD42b expression in HEL cells.

[0038] Figure 22Statistical analysis of alder ketone intervention on CD41 / CD42b expression in HEL cells (*Compared with the control group, *P<.05, **P<.01, ***P<.001).

[0039] Figure 23 Figure showing the effect of different concentrations of alder ketone on the cytotoxicity of Meg-01 cells.

[0040] Figure 24 The figure shows the effect of different concentrations of alder ketone on the cytotoxicity of HEL cells. Detailed Implementation

[0041] This pharmaceutical composition can be prepared according to methods known in the art. It can also be prepared by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0042] Example 1: Preparation of oral tablets of alder ketone

[0043] Alderone: 20mg / tablet, Microcrystalline cellulose (filler): 55mg / tablet, Sodium carboxymethyl starch (disintegrant): 23mg / tablet, Magnesium stearate (lubricant): 2mg / tablet, Total tablet weight: Approximately 100mg / tablet.

[0044] Alder ketone, microcrystalline cellulose, sodium carboxymethyl starch, and magnesium stearate are mixed thoroughly. Alder ketone is then added to the mixed excipients and stirred until homogeneous. Wet granulation or dry granulation is used to improve the flowability of the material, facilitating subsequent tableting. Particles obtained from wet granulation need to be dried to remove excess moisture, ensuring granule stability and flowability. The dried granules are fed into a tableting machine and compressed into tablets. Pressure is controlled during tableting to ensure tablet hardness and disintegration properties. The prepared tablets undergo quality checks, including content, dissolution rate, disintegration time, and appearance, to ensure compliance with specified quality standards. The qualified tablets are then packaged, typically using blister packs, bottles, or aluminum foil / polyethylene composite film.

[0045] Example 2: Preparation of oral syrup of alder ketone

[0046] Alderone: 2%, Sucrose: 50%, Lactose: 10%, Water: as needed, balance to 100%. Add sucrose to an appropriate amount of water, heat to 80-90℃, and stir until completely dissolved to make a syrup. Add lactose: Add lactose to the syrup and stir well until completely dissolved. Add alderone to the syrup and stir well. Adjust the pH to 5.0-7.0 using sodium hydroxide or hydrochloric acid solution for oral administration. Add purified water to the prescribed volume and stir well. Filter the mixture to remove insoluble impurities. Check the syrup's pH, drug content, stability, etc., to ensure it meets quality standards. Fill the qualified syrup into appropriate containers.

[0047] Example 3: Preparation of oral granules of alder ketone

[0048] Weigh alfalfa ketone: 20 mg / g, microcrystalline cellulose (filler): 50 mg / g, sodium carboxymethyl starch (disintegrant): 10 mg / g, and magnesium stearate (lubricant): 2 mg / g. Mix the alfalfa ketone and excipients thoroughly, ensuring uniform material distribution. Press the mixture into granules using a dry granulator. Sieve the granules through a sieve to remove powder and defective particles. Check the granule content, appearance, particle size, and other indicators to ensure they meet quality standards.

[0049] The following pharmacodynamic tests demonstrate the beneficial effects of this invention.

[0050] Example 1: In vivo pharmacodynamic study of alnustone in mice with radiation-induced thrombocytopenia

[0051] Alder ketone source: The natural monomer of alder ketone was purchased from Chengdu Pufeed Biotechnology Co., Ltd., product number JOT-10698, purity ≥98%, CAS: 33457-62-4, molecular weight: 262.352.

[0052] 1. Experimental Methods

[0053] ① Grouping and Modeling: Sixty 6-8 week old Kunming mice, half male and half female, were used. After 7 days of acclimatization, 10 mice were reserved as a control group without irradiation. The remaining mice were irradiated with X-rays (4 Gy) all over their bodies. On the first day after irradiation, blood was collected from the fundus venous plexus of the mice. The blood routine levels were detected by a Sysmex XT-2000iV hematology analyzer. Based on the baseline levels of PLT, RBC, and WBC, the irradiated model mice were randomly divided into the model group (irradiated only, without drug administration), the positive drug TPO group, the low-dose alderne group (5 mg / kg, denoted as ALN-L), the medium-dose alderne group (10 mg / kg, denoted as ALN-M), and the high-dose alderne group (20 mg / kg, denoted as ALN-H).

[0054] ② Drug preparation and administration method

[0055] TPO preparation method: Take 72 μL of 15000 U / mL TPO, dilute it with physiological saline to 4 mL, store on ice protected from light, and use immediately. Alderne preparation method: Take 36 mg of alderne monomer powder, dissolve it in 6 mL of 1% Tween 80 to prepare a 6 mg / mL working solution, and use immediately.

[0056] The unirradiated control group and the irradiated model group were injected intraperitoneally with 0.1 mL of physiological saline per 10 g of mouse body weight. The positive control drug TPO group was converted to 2700 U / kg based on the human (3000 U / kg) and mouse dose ratio. The low-dose alderne group (ALN-L) was administered intraperitoneally at 5 mg / kg / day, the medium-dose alderne group (ALN-M) at 10 mg / kg / day, and the high-dose alderne group (ALN-H) at 20 mg / kg / day. All drug groups were injected at a volume of 0.1 mL / 10 g. Intraperitoneal injections were administered once daily from day 1 after blood collection (day 2 after irradiation) until day 12. From the start of the modeling process until the end of the experiment, the mice's activity, mental state, coat luster, and mortality were observed daily. Mouse weight was measured every 3 days, and medication was administered based on the updated weight.

[0057] ③ Sample Collection and Detection Indicators: On days 1, 4, 7, 10, and 12 after mouse irradiation, 40 μL of blood from the orbital venous plexus was collected and added to EP tubes pre-filled with 160 μL of diluent. The mixture was gently pipetted and stirred. Peripheral PLT and MPV levels were analyzed using a Sysmex XT-2000iV hematology analyzer. On day 12, mice were euthanized by cervical dislocation. The liver, spleen, kidney, and thymus were removed, weighed, and the organ index was calculated: Organ Index = Organ Weight (kg) / Mouse Body Weight (kg). The femur of the mice was removed, fixed with 4% paraformaldehyde, and the number of megakaryocytes in the bone marrow, the main hematopoietic tissue, was observed using H&E staining. Bone marrow cells were collected from the femur of the mouse and centrifuged at 1500 rpm for 5 min at 4°C. The cells were resuspended in PBS and CD41 and CD61 flow cytometry antibodies were added. The cells were incubated in the dark for 30 min. The expression of CD41 / CD61 in bone marrow, spleen and lung megakaryocytes was detected and analyzed by flow cytometry.

[0058] 2. Experimental Results

[0059] Experimental Results: As shown in Table 1, the changes in platelet levels in mice revealed that platelet counts decreased rapidly after irradiation, initially at a slower rate than in the irradiation group. Platelet counts reached their lowest point on day 7 post-irradiation, but the positive control group (TPO), alder ketone group (5 mg / kg, 10 mg / kg, 20 mg / kg, denoted as ALN-L, ALN-M, ALN-H, respectively) showed higher platelet levels than the model group (*Compared with the model group, *P<.05, **P<.01, ***P<.001). Subsequently, platelet levels gradually recovered, with the alder ketone group recovering more rapidly than the model group. On day 12 post-irradiation, the positive control group (TPO) and alder ketone group showed the highest platelet counts. (5 mg / kg, 10 mg / kg, 20 mg / kg, representing ALN-L, ALN-M, ALN-H respectively) significantly increased (*Compared with the model group, *P<.05, **P<.01, ***P<.001); By day 13, platelet counts had basically returned to normal levels, and there were significant differences between the alder ketone treatment group and the model group (*Compared with the model group, *P<.05, **P<.01, ***P<.001); As shown in Table 2, the mean platelet volume (MPV) of mice did not change significantly. Tables 1 and 2 show that alder ketone has a significant effect on platelet recovery, indicating that alder ketone has a significant therapeutic effect on irradiated thrombocytopenia mice.

[0060] Table 1. Effects of alnustone on platelet production in mice (*Compared with the model group, *P<.05, **P<.01, ***P<.001)

[0061]

[0062] Table 2. Effects of alnustone on mean platelet volume (MPV) in mice.

[0063]

[0064] according to Figure 1 (Platelet count results) and Figure 2 (MPV statistics) show that: ( Figure 1Compared with the model group, the platelet levels in mice treated with alfalfa (5 mg / kg, 10 mg / kg, 20 mg / kg, denoted as ALN-L, ALN-M, ALN-H, respectively) and the positive drug TPO group significantly increased after day 7 of modeling. Although they did not recover to the levels of the TPO group, the alfalfa groups (ALN-L, ALN-M, ALN-H) showed a significant effect in restoring platelet levels (*Compared with the model, *P<.05, **P<.01, ***P<.001), indicating that alfalfa has a certain therapeutic effect on irradiated thrombocytopenic mice and a significant effect in restoring platelet levels. Figure 2 Furthermore, alfalfa ketone restored platelet levels without significantly altering mean platelet volume (*Compared with the model, *P<.05, **P<.01, ***P<.001), indicating that alfalfa ketone administration can promote platelet recovery without affecting mean platelet volume.

[0065] according to Figure 3-8 It can be known that: Figure 3 , Figure 5 , Figure 7 (Results image) and Figure 4 , Figure 6 , Figure 8 (Statistical analysis chart) shows that the FITC-CD41(+)-PE-CD61(+) positive areas represent the double-positive areas of CD41 and CD61, surface markers for megakaryocyte differentiation and maturation in bone marrow, spleen, and lung. (CD41 is a specific surface antigen of megakaryocytes, expressed throughout the entire process of megakaryocyte differentiation; CD61 is a specific surface antigen of megakaryocytes, and its expression tends to be the earliest expressed glycoprotein in megakaryocytes, a single-chain surface membrane glycoprotein). Compared with the model group, in vivo administration of gradient concentrations of alderone (5 mg / kg, 10 mg / kg, 20 mg / kg, represented as ALN-L, ALN-M, ALN-H, respectively) on day 12 significantly promoted the expression of surface antigens CD41 / CD61 during megakaryocyte differentiation, indicating that alderone can promote megakaryocyte differentiation in bone marrow, spleen, and lung. Figure 4 , Figure 6 and Figure 8 )right Figure 3 , Figure 5 and Figure 7Statistical analysis of flow cytometry results showed that after 12 days of in vivo administration of alfalfa (5 mg / kg, 10 mg / kg, 20 mg / kg), the expression rates of CD41(+) / CD61(+) in bone marrow, spleen, and lung cells of the alfalfa-treated groups (5 mg / kg, 10 mg / kg, 20 mg / kg) were significantly higher than those of the model group on day 12, approaching those of the normal control group. The differentiation of pulmonary megakaryocytes was statistically significant (*P<.05, **P<.01, ***P<.001 compared with the model group), indicating that in vivo administration of alder ketone not only restored platelet levels but also significantly stimulated the expression of CD41 / CD61 surface antigens on megakaryocytes in the bone marrow, spleen, and lungs. Therefore, alder ketone has a significant promoting effect on megakaryocyte differentiation in the bone marrow, spleen, and lungs, suggesting that the hematopoietic effect of alder ketone is mainly concentrated in bone marrow, spleen, and lung hematopoiesis.

[0066] according to Figure 9-12 It can be known that: Figure 9 , Figure 11 (Results image) and Figure 10 , Figure 12 (Statistical analysis chart) shows that on day 12 of in vivo administration of alder ketone, compared with the model group, the alder ketone administration groups (5 mg / kg, 10 mg / kg, 20 mg / kg, represented as ALN-L, ALN-M, ALN-H, respectively) and the positive control group TPO significantly increased the size and number of megakaryocytes in the bone marrow, spleen, and lung. The morphology and lobulation of megakaryocytes were not significantly different from the normal control group, indicating that alder ketone administration promoted the restoration of normal megakaryocytes in the bone marrow, spleen, and lung of hematopoietic tissues. Simultaneously, alder ketone promoted megakaryocyte proliferation and differentiation. H&E staining of megakaryocytes in the bone marrow, spleen, and lung of the normal control group, model group, positive control group TPO group, and alder ketone administration group further confirmed this effect. Nucleated cells were counted, and statistical analysis revealed a significant increase in megakaryocytes in the bone marrow, spleen, and lungs treated with alfalfa (5 mg / kg, 10 mg / kg, 20 mg / kg, denoted as ALN-L, ALN-M, ALN-H, respectively). Although the effect was not as significant as that of the positive control drug TPO group, the number of megakaryocytes was significantly higher than that in the model group. Furthermore, with increasing drug concentration, the number of megakaryocytes in the bone marrow, spleen, and lungs tended to return to normal levels, indicating that alfalfa promoted the proliferation and differentiation of megakaryocytes in the bone marrow, spleen, and lung tissues, thus confirming the above flow cytometry results (*Compared with the model group, *P<.05, **P<.01, ***P<.001).

[0067] Experimental Example 2: Effect of Alnustone on Platelet Production in Transgenic Zebrafish

[0068] 1. Experimental Methods

[0069] The promoter of the zebrafish CD41 gene was cloned and ligated into the pEGFP-1 vector via enzyme digestion. A 6kb DNA sequence containing the CD41 promoter was identified and cloned in the pEGFP-1 expression vector and placed upstream of the GFP cDNA to form the CD41-GFP structure. The CD41-GFP plasmid was double-digested with XhoI and NaeI, and the restriction fragments were separated by 0.7% agarose gel electrophoresis. The DNA fragments were purified using the Compass DNA purification kit. The recovered DNA was extracted once with phenol / chloroform, precipitated with ethanol, and resuspended in 150mM KCI (50-100 ng / L) with 0.1% phenol red. Using standard microinjection techniques, approximately 30-50 pg of DNA was microinjected into multiple zebrafish embryos at the single-cell stage to construct a CD41-GFP transgenic zebrafish model.

[0070] CD41-GFP transgenic zebrafish preserved in a water cycle were used in the experiment. The experiment included a control group (with cells, without alderne, with culture medium, 3 replicates) and alderne-treated groups (with cells, dosages: 10 μM, 20 μM, 40 μM, with culture medium, 3 replicates for each dosage). Five-day-old CD41-GFP transgenic zebrafish larvae were anesthetized with gradient concentrations of alderne (10 μM, 20 μM, 40 μM) and then observed under a laser confocal microscope to observe the effect of alderne on platelet production.

[0071] 2. Experimental Results

[0072] Experimental results are as follows Figure 13-14 As shown.

[0073] according to Figure 13 (Results image) and Figure 14 (Statistical analysis chart) shows that: ( Figure 13 Compared with the control group, the number of GFP-labeled platelets (concentrated in the zebrafish tail) in the alderne (ALN) treatment groups (10 μM, 20 μM, 40 μM) was significantly increased, and an increase in GFP-labeled platelet spots could be clearly seen in the dorsal aorta and tail hematopoietic tissue; Figure 14 )right Figure 13Statistical analysis of platelet fluorescent spots revealed that the average number of fluorescent spots in the control group was 30.67±2.52, while the average number of fluorescent spots in the alder ketone groups (10μM, 20μM, 40μM) were 47.33±4.04, 57.33±4.51, and 59.67±5.51, respectively. This indicates that alder ketone administration significantly increased the expression of GFP-labeled platelets in transgenic zebrafish, with statistically significant differences (*Compared with the control group, *P<.05, **P<.01, ***P<.001), suggesting that alder ketone promotes platelet production in zebrafish.

[0074] Experimental Example 3: Effects of Alnustone on Megakaryocyte Differentiation and Maturation

[0075] 1. Experimental Methods

[0076] Meg-01 and HEL cells were cultured in an incubator at 37°C and 5% CO2 using a basal medium containing 10% FBS, 1% penicillin-streptomycin mixture and RPMI-1640. The medium was changed daily. Once the cell density reached 80-90%, the cells were passaged at a ratio of 1:2.

[0077] Take healthy Meg-01 and HEL cells into 15 mL sterile centrifuge tubes, wash and resuspend them in phosphate-buffered saline (PBS), and then take 10 μL of the cell suspension for counting on a counting chamber. Adjust the cell density to 2 × 10⁻⁶ cells / cells. 4 cells / mL, at 2×10 4 Cells were seeded per well in 12-well plates at 500 μL per well. Experimental setups included a control group (cells present, no alderne, with culture medium, 3 replicates) and alderne treatment groups (cells present, drug concentrations: 10 μM, 20 μM, 40 μM, with culture medium, 3 replicates for each concentration). 500 μL of drug-containing culture medium was added to the alderne treatment groups, and the same amount (500 μL) was added to the control group. The final alderne concentrations were 10 μM, 20 μM, and 40 μM. Cells were incubated at 37°C in a 5% CO2 incubator using standard methods. After 5 days of incubation, the following experiments were performed:

[0078] ① Inverted microscope white light photography: Place the cells in a well plate and use an inverted microscope to magnify the cells 100 times to collect images on the 5th day of intervention, in order to observe the changes in cell morphology after drug administration and count the number of megakaryocytes.

[0079] ②Giemsa staining: Collect cells treated with gradient concentrations of alder ketone, centrifuge at 1500 rpm for 5 min, resuspend in pre-cooled PBS solution, add an equal volume of 0.075 mol / L KCl solution for swelling, centrifuge at 1500 rpm for 2 min, fix cells with fixative (methanol: glacial acetic acid = 3:1), and then perform Giemsa staining to observe the morphological changes of cells after treatment with gradient concentrations of alder ketone.

[0080] ③ Phalloidin staining: Collect cells treated with gradient concentrations of alfalfa ketone, centrifuge at 1500 rpm for 5 min, discard the supernatant, retain the cell pellet, wash once with PBS, fix with 4% paraformaldehyde for 10 min, and spin-slide. Permeabilize with 0.5% Triton X-100 solution for 5 min, wash three times with PBS; cover cells with diluted phalloidin working solution, incubate at room temperature in the dark for 1 h, wash three times with PBS; counterstain cell nuclei with DAPI for 20 s, wash three times with PBS; after mounting, use an inverted microscope to magnify the cells 100 times and acquire images.

[0081] 2. Experimental Results

[0082] Experimental results are as follows Figure 15-18 As shown.

[0083] ①According to Figure 15 (Results) As shown in the figure, compared with the control group, after intervention with gradient concentrations of alder ketone on day 5, alder ketone (10μM, 20μM, 40μM) all showed the same effect of promoting megakaryocyte differentiation, specifically manifested as a significant increase in megakaryocyte-like large cells (arrow), indicating that alder ketone has the potential activity of promoting megakaryocyte differentiation.

[0084] ②According to Figure 16 (Results) As shown in the figure, Giemsa staining results showed that on day 5, the control group had obvious mononuclear cells (the light color represents the cell membrane and the dark color represents the cell nucleus). However, compared with the control group, the alder ketone administration group (10μM, 20μM, 40μM) showed significant differences in the lobed state of the cell nuclei. The cells all showed a state of multiple lobed nuclei, and the number of multinucleated cells increased significantly. Among them, the high dose of alder ketone (40μM) even reached 16N or higher on day 5, indicating that alder ketone promotes the generation of polyploids during megakaryocyte differentiation.

[0085] ③According to Figure 17 and Figure 18(Results) As shown in the figure, the phalloidin staining results indicate that on day 5, compared with the control group, the alder ketone administration groups (10μM, 20μM, 40μM) significantly promoted the expression of multinucleated fluorescence in megakaryocytes (red light TRITCPhalloidin is located in the cell membrane microfilament structure, and blue light DAPI is located in the cell nucleus structure). Furthermore, the cell nuclei of the control group mostly showed a mononuclear state, while the cell nuclei of the administration groups showed a distinct multinucleated and lobulated state. This state was expressed on day 5 of alder ketone (10μM, 20μM, 40μM) intervention, and the multinucleated expression also showed an increasing trend with the increase of concentration, further indicating that alder ketone can promote the generation and maturation of polyploids during megakaryocyte differentiation.

[0086] Experiment 4: Investigation into the effect of alnustone on megakaryocyte differentiation

[0087] 1. Experimental Methods

[0088] Meg-01 and HEL cells were cultured in an incubator at 37°C and 5% CO2 using a basal medium containing 10% FBS, 1% penicillin-streptomycin mixture and RPMI-1640. The medium was changed daily. Once the cell density reached 80-90%, the cells were passaged at a ratio of 1:2.

[0089] Meg-01 and HEL cells in good growth condition were placed in 15 mL sterile centrifuge tubes, washed and resuspended in phosphate-buffered saline (PBS), and 10 μL of the cell suspension was counted on a counting chamber. The cell density was adjusted to 2 × 10⁻⁶ cells / cells. 4 cells / mL, at 2×10 4 Cells were seeded per well in 12-well plates at 500 μL per well. Experimental setups included a control group (cells present, no alderne, with culture medium, 3 replicates) and alderne treatment groups (cells present, drug concentrations: 10 μM, 20 μM, 40 μM, with culture medium, 3 replicates for each concentration). 500 μL of drug-containing culture medium was added to the alderne treatment groups, and the same amount (500 μL) was added to the control group. The final alderne concentrations were 10 μM, 20 μM, and 40 μM. Cells were incubated at 37°C in a 5% CO2 incubator using standard methods. After 5 days of incubation, the following experiments were performed:

[0090] Flow cytometry detection of CD41 / CD42b expression: Cells were collected, centrifuged at 1500 rpm for 5 min, washed twice with pre-cooled PBS, mixed with 100 μL of PBS, centrifuged at 1500 rpm for 5 min, and the supernatant was aspirated. 3 μL of LCD41 antibody and 3 μL of LCD42b antibody were added, and the cells were placed on ice in the dark for 15 min. Before sample injection, 200-400 μL of PBS was added, and the cells were detected at an excitation wavelength of 488 nm to detect the expression of the surface markers CD41 / CD42b during megakaryocyte differentiation.

[0091] 2. Experimental Results

[0092] Experimental results are as follows Figure 19-22 As shown.

[0093] according to Figure 19 , Figure 20 (Results image) and Figure 21 , Figure 22 (Statistical analysis chart) shows that the FITC-CD41(+)-PE-CD42b(+) positive area represents a double-positive area of ​​CD41 and CD42b, surface markers for megakaryocyte differentiation and maturation (CD41 is a specific surface antigen of megakaryocytes, expressed throughout the entire process of megakaryocyte differentiation; CD42b is a specific surface antigen of megakaryocytes, and its expression tends to be more differentiated and mature megakaryocytes, indicating terminal maturation of the megakaryocyte lineage). Compared with the control group, the intervention with gradient concentrations of alfalfa (10μM, 20μM, 40μM) on day 5 can significantly promote megakaryocyte differentiation and maturation. The expression of surface antigens CD41 / CD42b during differentiation indicated that aldosterone at concentrations of 10 μM, 20 μM, and 40 μM could promote megakaryocyte differentiation. Statistical analysis of flow cytometry results showed that the expression rates of CD41(+) / CD42b(+) in Meg-01 and HEL cells treated with aldosterone were significantly increased, which was statistically significant (*Compared with the control group, *P<0.05, **P<0.01, ***P<0.001); indicating that aldosterone can significantly stimulate the expression of surface antigens CD41 / CD42b in Meg-01 and HEL cells.

[0094] Experimental Example 5: Study on the cytotoxicity of alnustone

[0095] 1. Experimental Methods

[0096] Meg-01 and HEL cells were cultured in an incubator at 37°C and 5% CO2 using a basal medium containing 10% FBS, 1% penicillin-streptomycin mixture and RPMI-1640. The medium was changed daily. Once the cell density reached 80-90%, the cells were passaged at a ratio of 1:2.

[0097] Cytotoxicity assay: Healthy Meg-01 and HEL cells were placed in 15 mL sterile centrifuge tubes, washed and resuspended in phosphate-buffered saline (PBS), and 10 μL of the cell suspension was counted on a counting chamber. The cell density was adjusted to 2 × 10⁻⁶ cells / cells. 4Cells / mL were seeded at 4000 cells / well in 96-well plates, 180 μL per well. Experimental setups included a background group (no cells, no drug, only culture medium), a drug-containing background group (no cells, containing drug and culture medium), a control group, and alderne treatment groups (10 μM, 20 μM, 40 μM). 20 μL of drug-containing culture medium was added to the alderne treatment groups and the drug-containing background group, while 20 μL of drug-free culture medium was added to the control group and the background group. Each group was divided into three replicates and cultured routinely at 37°C in a 5% CO2 incubator. After culturing for 2, 4, and 6 days, cell proliferation rate was detected using the CCK8 assay (Cell Counting Kit-8, Dojindo, specification: 100mL / bottle, batch number: LZ735). The experiment was performed in three independent replicates: 20μL of CCK8 solution was added dropwise to each well, mixed, and incubated for 1 hour. The absorbance (OD) value was then measured at 490nm. The final OD value was calculated as: Detected value - Background group - Drug-containing background group.

[0098] 2. Experimental Results

[0099] Experimental results are as follows Figure 23 and Figure 24 (Statistical analysis chart) As shown, after intervention with 2.5μM, 5μM, 10μM, 20μM, and 40μM alderne in Meg-01 and HEL cells, respectively, compared with the control group, the cell survival rates at each time point were comparable to those in the control group after intervention with alderne (2.5μM, 5μM, 10μM, 20μM, 40μM) for 2, 4, and 6 days, with no significant difference. Cytotoxicity experiments demonstrated that alderne is safe for treating thrombocytopenia.

[0100] In summary, this invention provides a new use for alnustone, namely, its application in the preparation of drugs for treating thrombocytopenia. In vitro experiments have demonstrated that when Meg-01 cells and HEL cells were treated with alnustone on days 2, 4, and 6, the cell survival rates of the 10 μM, 20 μM, and 40 μM alnustone-treated groups were comparable to those of the control group at each time point, and the 10 μM, 20 μM, and 40 μM concentrations of alnustone were not toxic to the cells. The number of megakaryocytes in the alder ketone treatment groups (10 μM, 20 μM, 40 μM) showed a significant increasing trend, indicating that alder ketone at concentrations of 10 μM, 20 μM, and 40 μM has the activity of promoting megakaryocyte differentiation, and this trend is concentration-dependent. On day 5 of alder ketone (10 μM, 20 μM, 40 μM) intervention in Meg-01 cells and HEL cells, the expression rate of CD41 / CD42b was significantly increased, which was statistically significant (*compared to the control group). The results (*P<.05, **P<.01, ***P<.001) indicate that alder ketone significantly stimulates the expression of CD41 / CD42b surface antigens in Meg-01 and HEL cells. Significant differences were observed in the nuclear lobulation state of cells in the alder ketone treatment groups (10 μM, 20 μM, 40 μM), with all cells exhibiting multinucleated lobulation and a significantly increased number of multinucleated cells, indicating that alder ketone promotes polyploid formation during megakaryocyte differentiation. On day 5 of alder ketone (10 μM, 20 μM, 40 μM) intervention in Meg-01 and HEL cells, polynucleated cell expression increased with increasing concentration, further demonstrating that alder ketone can promote polyploid formation and maturation during megakaryocyte differentiation. In vivo experiments in mice demonstrated that platelet levels in mice treated with alfalfa (5 mg / kg, 10 mg / kg, 20 mg / kg) and the positive control drug TPO group significantly increased after day 7 of modeling. Although the platelet levels did not return to those of the positive control drug TPO group, the alfalfa treatment groups (5 mg / kg, 10 mg / kg, 20 mg / kg) showed a significant effect in restoring platelet levels, indicating that alfalfa has a certain therapeutic effect on irradiated thrombocytopenia mice and a significant effect on restoring platelet levels. Furthermore, while restoring platelet levels, alfalfa did not significantly change the mean platelet volume, indicating that alfalfa administration can promote platelet recovery without affecting mean platelet volume. Compared with the model group, the expression rates of CD41 / CD61 in bone marrow cells, spleen cells, and lung cells were significantly increased on day 12 after in vivo administration of alder ketone, approaching the differentiation levels of bone marrow cells, spleen cells, and lung cells in the control group, which was statistically significant. This indicates that in vivo administration of alder ketone can not only restore platelet levels but also significantly stimulate the expression of CD41 / CD61 surface antigens in bone marrow cells, spleen cells, and lung cells. Therefore, alder ketone has a significant promoting effect on the differentiation of bone marrow cells, spleen cells, and lung cells, suggesting that the hematopoietic effect of alder ketone is mainly concentrated in bone marrow, spleen, and lung hematopoiesis.

Claims

1. Use of alder in the preparation of drugs for treating thrombocytopenia.

2. The use according to claim 1, characterized in that: The drug described is for treating thrombocytopenia caused by aplastic anemia.

3. The use according to claim 1, characterized in that: The drug described is a drug that restores platelet levels and promotes platelet production in patients with thrombocytopenia.

4. The use according to claim 1, characterized in that: The drug described is a drug that promotes the differentiation and maturation of megakaryocytes in the bone marrow, spleen, and lungs of hematopoietic tissue.

5. The use according to any one of claims 1-4, characterized in that: It is a pharmaceutical preparation made from an effective amount of alder ketone as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients.

6. The use according to claim 5, characterized in that: The pharmaceutical preparations mentioned are oral preparations, injectable preparations, topical preparations, sustained-release preparations, or controlled-release preparations.

7. The use according to claim 6, characterized in that: The oral preparations include capsules, granules, tablets, mixtures, or syrups.

Images