CN117122605A

CN117122605A - Application of myricetin in preparation of medicament for treating thrombocytopenia

Info

Publication number: CN117122605A
Application number: CN202310400773.8A
Authority: CN
Inventors: 吴建明; 邓君竹; 王龙; 黄飞鸿; 罗杰斯; 曾静; 吴安国; 周晓钢; 陈琪; 黄倩倩
Original assignee: Southwest Medical University
Current assignee: Southwest Medical University
Priority date: 2023-04-14
Filing date: 2023-04-14
Publication date: 2023-11-28

Abstract

The invention relates to the field of biological medicine, in particular to application of myricetin in preparation of medicines. In particular to the application of myricetin in the medicine for treating thrombocytopenia. Myrican is called pentahydroxyflavone-3-rhamnoside, widely existing in Yang Meide root bark, and is a flavonoid compound. The myricetin is mainly used for treating diabetes, cardiovascular diseases and the like, and through a great deal of experimental researches by the inventor, the myricetin is found to have no influence on the cell viability of megakaryocytes in vitro, has the effects of promoting the differentiation and maturation of megakaryocytes in bone marrow of main hematopoietic tissues and the generation of polyploids in vivo, and can promote the generation of blood platelets and restore the blood platelet level of patients with thrombocytopenia.

Description

Application of myricetin in preparation of medicament for treating thrombocytopenia

Technical Field

The invention relates to the field of biological medicine, belongs to novel application of micromolecular compound in pharmacy, and in particular relates to application of myricitrin in pharmacy, in particular to application in medicines for treating thrombocytopenia.

Background

Malignant tumors have become one of the public problems worldwide, severely threatening human health. Treatments for malignant tumors include surgical treatment, radiotherapy, chemotherapy, biological genetic engineering treatment, and the like, and scientists have developed a variety of different treatments in order to reduce the harm of malignant tumors to the life and health of patients. But the technical development is slow except for operation treatment, radiotherapy and chemotherapy. Radiotherapy is still one of the main methods for treating cancers, and although technology is advancing, toxic and side effects of radiotherapy still severely restrict treatment of malignant tumors.

Thrombocytopenia is the most common complication of radiotherapy, no specific medicine aiming at thrombocytopenia caused by radiotherapy exists in clinic at present, and small doses of recombinant human interleukin-11 (rh IL-11), recombinant human thrombopoietin (rhTPO) or platelet infusion are generally adopted. It is generally recommended that the administration of thrombopoietin be initiated after chemotherapy, once daily subcutaneously, for 7-10 days in succession. However, the platelet growth factor effect is often not very desirable and there is time lag, while the occurrence of severe risk of thrombocytopenia is small, usually with severe risk for only a short duration, and therapeutic targeting is not very good.

Yet another treatment is to infuse platelets into a patient, which can be used to quickly ameliorate the problem of thrombocytopenia in the patient by directly infusing platelets into the patient. However, the problem of platelet infusion iatrogenic infections is prominent and platelet acquisition is limited to the total amount of platelets that can be routinely obtained at a donor site by donor donation.

For the above drugs, thrombopoietin receptor agonist (TPO-RA) is usually selected. TPO-RA is a recommended thrombopoietic drug by the American blood Association, but is expensive, has a large number of side effects, cannot continuously act, and limits clinical use.

Therefore, there is a need for a therapeutic regimen or pharmaceutical compound that is effective against thrombocytopenia, or that is capable of preventing thrombocytopenia, for which the treatment of thrombocytopenia is not yet stable and reliable.

Disclosure of Invention

The invention aims to overcome the defects that the medicines for treating thrombocytopenia complicated with radiotherapy are expensive and lack a stable and reliable treatment means in the prior art, and provides a novel application of Myricetin (MYR), and provides a brand new medicine with low cost and small side effect for treating thrombocytopenia caused by radiotherapy.

In order to achieve the above object, the present invention provides the following technical solutions:

the application of myricitrin in preparing medicine for treating thrombocytopenia.

Myrican is called pentahydroxyflavone-3-rhamnoside, widely existing in Yang Meide root bark, and is a flavonoid compound. The myricetin is mainly used for treating diabetes, cardiovascular diseases and the like, and has no research on the therapeutic effect and the action mechanism of the myricetin on thrombocytopenia caused by radiotherapy so far.

Through a great number of experimental researches by the inventor, the myricetin has no influence on the cell viability of megakaryocytes in vitro, and has certain activity of promoting differentiation and maturation and generation of procalcitonin on megakaryocytes in vitro. Meanwhile, the research shows that the myricetin has the effect of promoting the generation of the blood platelets in vivo, can restore the blood platelet level of patients with the thrombocytopenia, and promotes the differentiation and maturation of megakaryocyte in bone marrow of main hematopoietic tissues and the generation of polyploid.

In vitro experiments prove that the large cells of the myricetin administration group (10 mu M,20 mu M and 40 mu M) obviously show an increasing trend, which shows that the myricetin at the concentration of 10 mu M,20 mu M and 40 mu M has activity for promoting megakaryocyte differentiation, and shows a concentration-dependent trend, and when the myricetin (10 mu M,20 mu M and 40 mu M) intervenes in K562 and Meg-01 cells on day 5, the expression rate of megakaryocyte markers CD41/CD42b is obviously increased, and the myricetin has statistical significance (P is less than.05, P is less than.01, P is less than.001), which shows that the myricetin can obviously stimulate the expression of the CD41/CD42b of the cell surface antigens of K562 and Meg-01; the cell nucleus split states of the positive medicine PMA (PKC activator, phorbol myristate acetic acid) group and the myricetin administration group (10 mu M,20 mu M and 40 mu M) are obviously different, the cells all show a plurality of nuclear split states, the nuclear split states of the myricetin administration group (10 mu M,20 mu M and 40 mu M) are similar to the nuclear split states of the positive medicine PMA group, and the number of polynuclear cells is obviously increased, so that the myricetin can promote the generation and maturation of polyploids in the process of megakaryocyte differentiation.

Further, the medicine is a medicine for treating thrombocytopenia caused by radiotherapy.

Further, the medicine is a medicine for treating thrombocytopenia caused by chemotherapy.

Further, the medicine also contains pharmaceutically acceptable auxiliary materials and/or carriers.

Furthermore, the daily dosage of the myricetin is within the range of 10-200mg. Preferably 10-100mg.

Or the content of myricetin in the medicine is 10-200mg. Preferably 10-100mg.

Furthermore, the daily dosage of the myricetin is in the range of 12-50mg.

Or the content of myricetin in the medicine is 12-50mg.

In vivo experiments of mice prove that the platelet level of the myricitrin administration group (2.5 mg/kg,5mg/kg,10 mg/kg) and the positive drug rhTPO administration group mice is obviously increased after 10 days of modeling, and the myricitrin administration group (10 mu M,20 mu M and 40 mu M) has obvious effect of restoring the platelet level although not restoring to the level of the positive drug TPO administration group, which indicates that the myricitrin has a certain treatment effect on irradiation induced thrombocytopenia mice and obvious effect of restoring the platelet level.

The daily dosage of myricetin for human body is converted to 14-60mg by the dosage of the mouse. The range of the pharmaceutical preparation can be expanded to 10-100mg, and the content of the active ingredients in the pharmaceutical preparation can be adjusted according to the subsequent human body test and research conditions.

Compared with a model group, the in vivo administration of myricetin has the advantages that the platelet level obviously returns on the 12 th day, the expression rate of CD41/CD61 and CD41/CD1117 in bone marrow cells is obviously increased, meanwhile, the myricetin can obviously promote the expression of CD41/CD61 in spleen cells, the in vivo administration of myricetin is similar to the bone marrow megakaryocyte differentiation condition of mice in a control group, the in vivo administration of myricetin has statistical significance, the in vivo administration of myricetin can not only restore the platelet level, but also can obviously stimulate the expression of bone marrow and spleen megakaryocyte surface antigens CD41/CD61 and megakaryocyte CD41/CD1117, so that the myricetin has obvious promotion effect on megakaryocyte differentiation in bone marrow, and the hematopoietic effect of myricetin is mainly shown to be similar to bone marrow, and spleen hematopoiesis can be promoted. Therefore, the myricitrin can be used for preparing medicines for treating thrombocytopenia caused by radiotherapy, and has a certain effect on treating thrombocytopenia caused by radiotherapy. Can restore the platelet level of patients suffering from thrombocytopenia caused by radiotherapy.

Cytotoxicity experiments show that on day 5, the myricetin is interfered with K562 and Meg-01 cells, and the cell activities of 10 mu M,20 mu M and 40 mu M myricetin administration groups are equivalent to those of cells of a control group at each time point, so that the myricetin administration groups have no toxicity to the cells and have small side effects. And the myricetin is widely existing in natural traditional Chinese medicinal materials, has low cost and great application potential for treating thrombocytopenia, and can be used for developing novel medicines for treating thrombocytopenia caused by radiotherapy.

Furthermore, myricetin achieves the effect of recovering the platelet number by promoting megakaryocyte differentiation and maturation and polyploid generation.

Furthermore, myricetin activates IGF1R/PI3K/AKT/GSK3 beta pathway by promoting phosphorylation of PI3K, akt, GSK3 beta protein, thereby promoting thrombopoiesis.

Further, the dosage form of the medicine is one of a tablet, a capsule and a pill.

Further, the drug sustained-release preparation or the controlled-release preparation.

Further, other active ingredients that promote thrombopoiesis are included in the medicament.

Further, the application in the preparation of the myricetin medicine also comprises a thrombopoietin receptor agonist.

Further, the thrombopoietin receptor agonist is TPO-RA.

Furthermore, the medicine also comprises recombinant human thrombopoietin.

Further, the medicine also comprises recombinant human interleukin-11.

Compared with the prior art, the invention has the beneficial effects that:

a large number of experimental researches show that the myricetin has no influence on the cell viability in vitro and has certain activity of promoting differentiation and maturation and generation of the procalcitonin on megakaryocytes in vitro; meanwhile, research shows that myricitrin has a promoting effect on platelet generation in vivo, can restore the platelet level of a mouse with thrombocytopenia caused by irradiation, promotes the differentiation and maturation of megakaryocyte and the generation of megakaryocyte progenitor cells in bone marrow and spleen of main hematopoietic tissues, thus the myricitrin has a certain effect on treating thrombocytopenia, and the myricitrin is widely existing in natural traditional Chinese medicinal materials, and has low cost and small side effect.

Drawings

FIG. 1 is a graph showing the effect of myricetin on cell viability.

FIG. 2 is a photograph of a cell-under-the-lens morphology of myricetin.

FIG. 3 is a graph showing the results of myricitrin on nuclear polyploid.

FIG. 4A is a graph showing the effect of myricitrin on F-actin expression by K562 cells on DMS membrane formation.

FIG. 4B is a graph showing the effect of myricitrin on F-actin expression by Meg-01 cells on DMS membrane formation.

FIG. 5 is a graph showing the effect of myricitrin on cell CD41-CD42b expression.

Fig. 6 is a graph of the effect of myricitrin on platelet levels in radiation-induced thrombocytopenia mice (P <.05, P <.01, P <.001 compared to model group).

FIG. 7 is a graph showing the effect of myricetin bone marrow cell CD41-CD61 expression (P <.05, P <.01, P <.001 compared to model group).

Fig. 8 is a graph showing the effect of myricetin bone marrow cell CD41-CD117 expression (P <.05, P <.01, P <.001 compared to model group).

Fig. 9 is a graph of the effect of myricetin spleen cell CD41-CD61 expression (P <.05, P <.01, P <.001 compared to model group).

FIG. 10 is a photograph and an analytical chart of the results of detection of the effect of myricetin on PI3K/AKT signaling pathway-associated proteins.

Detailed Description

The present invention will be described in further detail with reference to test examples and specific embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following embodiments, and all techniques realized based on the present invention are within the scope of the present invention.

Description of the terms

CD41 recognizes the platelet membrane glycoprotein GPIIb (i.e., integrin αiib chain, which non-covalently binds to gpiia "integrin β3 chain" to form GPIIb/IIIa complex); CD61 recognizes the platelet membrane glycoprotein gpiia (integrin beta 3 chain); CD42b reacts with GPIb on megakaryocytes and platelets. All three antibodies are expressed on the plasma and membrane of primitive megakaryocytes. By detecting the platelet marker CD41, the level of platelets can be reflected.

The invention provides a new application of myricitrin, namely an application of myricitrin in preparing a medicament for treating thrombocytopenia. Experimental records during the course of the specific study are described in the examples below.

Example 1

Research on cell viability by myricetin (myrricetin, MYR)

Basal medium (Roswell Park Memorial Institute 1640 medium) containing 10% fetal bovine serum medium (FBS), 1wt.% penicillin-streptomycin mixture and RPMI-1640 was used. At 37℃with 5% CO ₂ Culturing Meg-01 cells and K562 cells in incubator, changing liquid every day until the cell density reaches 80-90%, and mixing the liquid according to the ratio of 1:2 subculturing.

Meg-01 cells in the logarithmic growth phase and K562 cells were counted and inoculated in an amount of 5000 cells/well, and the system of each well was 100. Mu.L.

The experiments set up a blank, MYR dosing group (10 μm,20 μm,40 μm), each group set up five duplicate wells. Then adding the complete culture medium solution containing the myricetin with different concentrations into a 96-well plate, adding 100 mu L of the complete culture medium solution without the myricetin into a blank control group, finally sealing edges by using Phosphate Buffered Saline (PBS), avoiding edge effect, gently shaking the well plate, and placing the well plate into a cell culture box for culturing for 72 hours.

After the intervention, 20 mu L of CCK-8 solution (Cell Counting Kit-8 cell activity detection kit liquid medicine) is added into each hole, and the mixture is stirred gently and mixed uniformly and then placed into an incubator for 2 hours of culture.

After the cultivation is finished, the 96-well plate is taken out, the wavelength of the enzyme label instrument is set to 450nm, and the absorbance OD value is detected by an upper machine. The cell viability calculation formula is as follows:

cell viability (%) = (absorbance OD value of drug group)/(absorbance OD value of blank group) ×100%.

The experimental results are shown in FIG. 1.

As can be seen from FIG. 1, when myricitrin (10. Mu.M, 20. Mu.M, 40. Mu.M) intervenes in K562 cells and Meg-01 cells on day 3, the myricitrin administration group showed comparable survival rates with Control group cells at various time points, indicating that the 10. Mu.M, 20. Mu.M, 40. Mu.M concentrations of myricitrin were not toxic to cells, and these three safe concentrations could be selected for subsequent experiments.

Example 2

Myrican (myrricetrin, MYR) in vitro megakaryocyte differentiation assay

With a basal medium containing 10% Fetal Bovine Serum (FBS), 1wt.% penicillin-streptomycin mixture and RPMI-1640 at 37℃with 5% CO ₂ Culturing Meg-01 cells and K562 cells in incubator, changing liquid every day until the cell density reaches 80-90%, and mixing the liquid according to the ratio of 1:2 subculturing.

Meg-01 cells in the logarithmic growth phase and K562 cells were counted and inoculated in 12-well plates at a rate of 2 ten thousand cells/well, 500. Mu.L per well.

Experimental setup:

control group (with cells, without myricetin, with medium, 3 multiple wells);

positive medicine PMA group (with cells, the PMA group containing positive medicine is added with 500 mu L of basal culture containing PMA, the final concentration of PMA is 2.5nM, and the basal culture contains culture medium and 3 compound holes);

myrican administration group (cell-containing, administration: 10. Mu.M, 20. Mu.M, medium, 3 multiplex wells).

Adding 500 μl of the drug-containing basal culture into the myricetin administration group, adding 500 μl of the basal culture into the control group, and adding 5% CO at 37deg.C ₂ Culturing in incubator. After 5 days of culture, the following experiments were performed:

1. white light photographed cell morphology change of inverted microscope

Cells were placed in well plates and were magnified 100-fold using an inverted microscope to capture 5-day-old images of the intervention to observe changes in cell morphology after dosing and count the number of large cells.

As shown in FIG. 2, the effect of myricetin on cell morphology was observed. Compared with the control group, after the myricitrin is interfered on the 5 th day, the myricitrin (10 mu M,20 mu M and 40 mu M) has the same effect of promoting megakaryocyte differentiation, and the effect is particularly shown by the remarkable increase (arrow) of megakaryocyte, so that the potential activity of the myricitrin for promoting megakaryocyte differentiation is shown.

Giemsa staining to observe the effect of myricitrin on megakaryocyte morphology

Collecting cells of myricetin dry prognosis, centrifuging at 1500rpm for 5min, re-suspending in pre-cooled PBS solution, adding 0.075mol/L KCl solution with equal volume for swelling, centrifuging at 1500rpm for 2min, fixing the cells with fixing solution (methanol: glacial acetic acid=3:1, v/v), and then performing Giemsa staining to observe morphological change of the cells after myricetin intervention.

As shown in FIG. 3, the effect of myricitrin on megakaryocyte polyploid was observed. The Giemsa staining results show that on day 5, the control group can see obvious mononuclear cells (wherein the pale color is cell membrane and the dark color is nucleus), the cell nucleus split states of the myricitrin administration group (10 mu M,20 mu M and 40 mu M) have obvious differences, the cells all show the states of a plurality of nuclear split leaves, and the number of polynuclear cells is obviously increased, so that the myricitrin promotes the generation of polyploids in the megakaryocyte differentiation process.

3. Observation of F-actin expression influence of myricitrin cell DMS film formation by phalloidin staining

Collecting cells after the myricetin is dry, centrifuging the cells at 1500rpm for 5min by a centrifuge, discarding supernatant, retaining cell sediment, washing once by PBS, fixing 4% paraformaldehyde for 10min, and throwing slices. Permeabilizing 0.5% TritonX-100 solution for 5min, and washing with PBS three times; covering cells with diluted phalloidin working solution, incubating for 1h at room temperature in dark place, and washing with PBS for three times; DAPI counterstained nuclei for 20s, washed three times with pbs; after sealing, the cells were magnified 100-fold using an inverted microscope to collect pictures.

As shown in FIGS. 4A and 4B, the effect of F-actin expression by the formation of DMS membrane in myricitrin cells was observed. The phalloidin can dye fibrous kinetin (F-actin) in cells, and has red color. F-actin plays an important role in megakaryocyte differentiation and platelet formation. The result of the Phalloidin staining (fig. 4A and 4B) shows that, on day 5, compared with the control group, the myricitrin administration group (10 μm,20 μm,40 μm) can significantly promote the expression of megakaryocyte multi-nuclear fluorescence (red light TRITC Phillidine is localized in the cell membrane microfilament structure, blue light DAPI is localized in the cell nuclear structure), and the nuclear DAPI staining of the control group is in a mononuclear state, and the nuclear DAPI staining of the administration group is in an obvious multi-nuclear split state, indicating that myricitrin can promote the generation and maturation of polyploids in the megakaryocyte differentiation process.

4. Flow cytometry detection of original expression of myricitrin on megakaryocyte surface resistance

Collecting cells, centrifuging at 1500rpm for 5min, washing with PBS (precooling) for 2 times, adding 100 μl of PBS, mixing, centrifuging at 1500rpm for 5min, sucking out supernatant, adding 3 μLCD41 antibody and 3 μLCD42b antibody, standing on ice for 15min in dark place, adding 200-400 μLPBS before sample injection, and detecting with 488nm excitation light wavelength, thereby detecting expression of surface marker CD41/CD42b in megakaryocyte differentiation process.

The experimental results are shown in FIG. 5, and the results of flow cytometry detection of CD41-CD42b show that megakaryocytes tend to differentiate into mature cells. FITC-CD41 (+) -PE-CD42b (+) positive region represents the surface markers CD41, CD42b double positive region of megakaryocyte differentiation maturation (CD 41 is a specific surface antigen of megakaryocyte expressed throughout megakaryocyte differentiation; CD42b is a megakaryocyte specific surface antigen whose expression tends to differentiate to a higher degree, more mature megakaryocyte whose expression is predictive of terminal maturation of megakaryocyte lineage).

Compared with the control group, the myricetin administration group (10 mu M,20 mu M,40 mu M) can obviously promote the expression of the surface antigen CD41-CD42b in the megakaryocyte differentiation process when interfering on the 5 th day, and has statistical significance. The myricetin can obviously stimulate the expression of the surface antigens CD41-CD42b of K562 cells and Meg-01 cells.

Example 3

In vivo pharmacodynamics study of myricetin (myrricetin, MYR) on irradiation-induced thrombocytopenia mice

1.1 mouse modeling and grouping

KM mice (kunming mice) were acclimatized for one week during which time they were free to drink. After one week, the mice except the blank group were transported to an affiliated hospital of the university of southwest medical science for X-ray irradiation at a dose of 4Gy. After irradiation, the number of platelets was reduced because of bone marrow suppressed mice. On the first day after molding, the orbital venous plexus of the mice was collected and its blood image level was measured using a hemocytometer. The mice with similar modeling damage degree are randomly divided into a model control group, a MYR high-dose group (10 mg/kg), a MYR medium-dose group (5 mg/kg), a MYR low-dose group (2.5 mg/kg) and a rhTPO (3000U/kg) group by comprehensively referencing the levels of leucocytes, platelets and erythrocytes. Mice not irradiated were set as normal control groups of 8, male and female halves.

1.2 mode of administration in mice

After the completion of the grouping, the mice were started to be dosed with the specific dosing schedule shown in table 1 for a dosing period of 12 days. The fundus venous plexus was collected at 40 μl on days 4, 7, 10, and 12, respectively, and diluted with blood cell dilution, and checked on machine. During the dosing period, mice were observed daily for status and feeding, and mice were subjected to weight statistics every 2 days. After the experiment is completed, eyeballs are picked up for blood taking, and the liver, spleen, kidney, thymus and femur of the mice are peeled off. The viscera are weighed in time, then part of the viscera and the thighbone are fixed in 4% paraformaldehyde, and the rest viscera are used for flow detection.

Table 1 mouse dosing regimen

1.3 detection of peripheral blood images of mice

During the administration period, peripheral blood image detection is carried out on days 4, 7, 10 and 12 respectively, and the specific method is as follows: the fundus venous plexus blood (40. Mu.L) was taken by capillary tube and rapidly added to an EP tube containing 160. Mu.L of blood cell dilution, gently mixed, and the peripheral blood image of the mouse was detected by full-automatic blood cell analyzer.

1.4 flow assay for bone marrow cell CD41-CD61 and CD41-CD117 expression

After successful molding of the mice, the myricetin R is dosed for 12 continuous days, after the dosing is finished, the mice are killed by cervical vertebra removal, the two-sided femur of the mice are dissected and taken out, bone marrow cells in the femur are flushed out by using 2mL of normal saline, the flushed bone marrow cells are filtered by using a 200-mesh nylon net to obtain a bone marrow cell suspension of the mice, and then the bone marrow cell suspension is added with streaming preservation solution and preserved at 4 ℃. At the time of use, the cells are counted, and the number of the sucked cells is 1×10 ⁶ The cells were pelleted, pre-chilled PBS was added, 1200r/min, and centrifuged for 5min. The supernatant was decanted, and about 100. Mu.L of liquid was retained and gently mixed. Adding FITC-anti-Mouse-CD41 antibody 0.5 mu L and PE-anti-Mouse-CD61 antibody 1.25 mu L, mixing well, avoiding light,incubate on ice for 15min. And finally transferring the mixture to a flow pipe, uniformly mixing, and detecting by a machine.

The same sample was prepared as above, and 0.5. Mu.L of FITC-anti-Mouse-CD41 antibody and 0.625. Mu.L of PE-anti-Mouse-CD117 antibody were added, mixed well, protected from light and incubated on ice for 15min. And finally transferring the mixture to a flow pipe, uniformly mixing, and detecting by a machine.

1.5 flow assay of spleen cell CD41-CD61 expression

After successful molding of the mice, myricetin administration was performed for 12 consecutive days, after the end of administration, the mice were sacrificed by cervical vertebra removal, spleens of the mice were dissected and taken out, the length was cut to about 1/3, spleen tissues were gently ground on a nylon mesh, washed with physiological saline, spleen cells were collected by extraction, placed in a 1.5mL centrifuge tube (EP tube), centrifuged at 1200rpm for 5min, the supernatant was decanted, red cell lysate was added for 1mL, lysed on ice for 10min, and centrifuged at 1200rpm for 5min. At the time of use, spleen cells were counted and 1X 10 were aspirated ⁶ The cells were centrifuged at 1200r/min for 5min with 1mL of pre-chilled PBS. The supernatant was decanted, leaving approximately 100. Mu.L of liquid, and gently mixed. 0.5 mu L of FITC-anti-Mouse-CD41 antibody and 1.25 mu L of PE-anti-Mouse-CD61 antibody are added, mixed uniformly, and incubated for 15min on ice in the absence of light. And finally transferring the mixture to a flow pipe, uniformly mixing, and detecting by a machine.

1. Experimental results

2.1 influence of myricetin on peripheral blood image of thrombocytopenia mice

After molding, administration was performed daily, blood image detection was performed at a specific time, and fig. 6 shows the result of platelet count detection, wherein the platelet count of mice was relatively uniform the first day after molding; on day 4 after mouse molding, platelet numbers compensatory rise due to radiation of radiation; on day 7, the model group mice had minimal platelet count, but the rhTPO group mice had a slowed decrease in platelet count (P < 0.01). At this time, the effect of each dose group of myricitrin on platelets is obvious (P > 0.05); on day 10, the number of platelets was significantly increased (P < 0.001) in each dose group of myricitrin and in the rhTPO group, but there were also differences compared to the mice in the blank group; on day 12, the number of platelets in the high, low dose and rhTPO groups of myricitrin was substantially leveled with the mice in the placebo group (P < 0.001). Table 2 is a record of the change in platelet count.

TABLE 2 influence of myricetin on mouse thrombopoiesis

In the table, P <.05, P <.01, P <.001 compared to model group.

2.2 Effect of myricetin on the expression of CD41-CD61 and CD41-CD117 in mouse bone marrow cells

After the end of the administration of the mice, the femur of the mice was dissected out to wash out bone marrow cells, and the expression of CD41-CD61 and the expression of CD41-CD117 of the bone marrow cells were examined by flow cytometry. As a result, as shown in FIG. 7, the expression of CD41-CD61 was significantly reduced in the bone marrow cells of the mice in the model group (P < 0.001) as compared with the mice in the normal control group, and the expression of CD41-CD61 was significantly increased in each of the myricetin dose group and the rhTPO group as compared with the mice in the model control group, and the difference was statistically significant (P < 0.001). As shown in FIG. 8, the expression of CD41-CD117 was significantly reduced in the bone marrow cells of mice in the model group (P < 0.001) compared to the normal control group mice, and the expression of CD41-CD117 was significantly increased in each of the dose group of myricetin and the rhTPO group (P < 0.001) compared to the model control group mice. The results show that myricitrin can restore bone marrow suppression and promote megakaryocytogenesis of mice with thrombocytopenia caused by radiation modeling.

2.3 Effect of myricetin on mouse spleen cell CD41-CD61 expression

The spleen is another important hematopoietic site other than bone marrow and lung, and when the hematopoietic function of bone marrow is impaired or lost, the spleen exerts extramedullary hematopoietic function to compensate for the deficiency of hematopoietic function of bone marrow. After the end of the administration of the mice, the spleens of the mice were removed, a portion of the spleens were cut off to extract spleen cells, and the expression of CD41-CD61 of the spleen cells was detected by flow cytometry. The results are shown in FIG. 9, in which the expression of CD41-CD61 in spleen cells was significantly reduced (P < 0.001) in mice in the model group as compared with mice in the normal control group, and the expression of CD41-CD61 was significantly increased (P <0.001, P < 0.01) in each of the dose group of myricetin and the rhTPO group as compared with the model control group. The result shows that the myricetin can promote the differentiation of spleen cells.

Through the cell experiments and the animal experiments in the embodiments 1-3, the myricitrin has the new application of promoting the generation of the blood platelet, namely, the myricitrin can be applied to the preparation of the medicine for treating the thrombocytopenia.

In the experimental investigation, the cell experiment proves that the myricetin has no influence on the cell viability of megakaryocytes in vitro and has certain activity of promoting differentiation and maturation and generation of the pre-platelets on the megakaryocytes in vitro. Animal experiments prove that the myricetin has the effect of promoting the generation of blood platelets in vivo, can restore the blood platelet level of patients with thrombocytopenia, and promotes the differentiation and maturation of megakaryocyte in bone marrow of main hematopoietic tissues and the generation of polyploid. Therefore, cell experiments and animal experiments confirm that myricetin has a certain effect on treating thrombocytopenia.

Example 4

Western Blot (Western Blot) was used to detect expression of Meg-01 cellular proteins.

Experiment setting: blank control group, myricetin 10 mu mol/L group, myricetin 20 mu mol/L group and myricetin 40 mu mol/L group.

Western blotting kit: commercially available product, brand Cell Signaling(CST)。

A stock solution of myricetin (50. Mu. Mol/L) was diluted with 10% complete medium to working solution and added to a 12-well plate at 500. Mu.L/well. K562 cells in the logarithmic growth phase, which were well-conditioned and clean in background color, were centrifuged at 600r/min for 5min, and the supernatant was removed, resuspended in 10% complete medium and counted. Inoculating K562 cells into the 12-well plate with plate density of 1×10 ⁴ cells/mL, 500. Mu.L of the cell suspension was added and mixed well.

Is placed at a relative humidity of 95%; CO ₂ Concentration 5%; culturing in a cell culture incubator at 37 ℃. On day 5 of the experiment, cells were removed, centrifuged at 1200rpm for 4min, the supernatant was aspirated off, and 200. Mu.L of RIPA cells were addedCracking the cracking solution on ice for 15min, and placing into a refrigerator at-80 ℃. On day 2 of cooling, protein concentration was measured as follows: and (3) sucking 2 mu L of protein sample, uniformly mixing the protein sample into 300 mu L of protein concentration measuring solution, taking 250 mu L of protein sample, adding the protein sample into a 96-well plate, reading the value by using a multifunctional enzyme-labeling instrument, and calculating the protein concentration of the sample.

Protein concentration of sample = [ (sample OD value-blank background OD value)/2-0.0168 ]/0.1127.

And (5) determining the loading amount, preparing glue, loading, electrophoresis and transferring films. After the transfer, the membrane was blocked with a rapid blocking solution and then washed three times with PBST (Tween-20-containing phosphate buffer solution) for 10min each time. The membrane was placed in the corresponding primary antibody (diluted 1:1000 fold), the primary antibody was recovered overnight, incubated with secondary antibody (diluted 1:2000 fold) and developed.

Finally, the ratio of the phosphorylated protein to the total protein gray value is analyzed and calculated by using ImageJ software.

As a result of experiments, the effect of PI3K/Akt signaling pathway on megakaryocyte differentiation and thrombopoiesis has been widely reported, and based on research on MYR megakaryocyte differentiation in the earlier stage of a subject group, and simultaneously, combining network pharmacology with pathway prediction on MYR for treating thrombocytopenia, we primarily judge that MYR action mechanisms are related to the PI3K/Akt signaling pathway. IGF-1R is a key target for network pharmacology prediction and can also serve as a hematopoietic factor, and is downstream of PI3K/AKT, so that MYR is predicted to activate IGF-1R/PI3K/AKT/GSK3 beta signaling pathway. The results are shown in FIG. 10, and compared with the blank control group, after MYR is interfered with Meg-01 cells, phosphorylation of PI3K, akt, GSK3 beta protein is obviously promoted, and IGF1R/PI3K/AKT/GSK3 beta pathway is activated. MYR was shown to exert an effect of promoting thrombopoiesis by activating the pathways described above.

Example 5

A pharmaceutical tablet contains myricetin as active ingredient and common tabletting adjuvants. Wherein, the dosage of myricetin is 10 mu M,20 mu M,40 mu M, 50 mu M and 100 mu M.

Example 6

A capsule contains myricetin as active ingredient and common capsule adjuvant. Wherein, the dosage of myricetin is 10 mu M,20 mu M,40 mu M, 50 mu M and 100 mu M.

Example 7

A suspension preparation contains myricetin as active ingredient and common suspension preparation adjuvant. Wherein, the dosage of myricetin is 10 mu M,20 mu M,40 mu M, 50 mu M and 100 mu M.

Example 8

A lyophilized injection contains myricetin and thrombopoietin receptor agonist as active ingredients.

Example 9

A lyophilized injection contains myricetin and recombinant human interleukin-11 as active ingredients.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. Use of myricitrin according to claim 1 for the preparation of a medicament for the treatment of thrombocytopenia.

2. The use of myricitrin according to claim 1 in the preparation of a medicament for the treatment of thrombocytopenia caused by radiation therapy.

3. The use of myricitrin according to claim 1 in the preparation of a medicament for the treatment of chemotherapy-induced thrombocytopenia.

4. The use of myricetin according to claim 1 in the preparation of a medicament, wherein the medicament further comprises pharmaceutically acceptable excipients and/or carriers.

5. The use of myricitrin according to claim 1 in the preparation of a medicament, wherein the daily dosage of myricitrin is in the range of 10-200mg.

6. The use of myricitrin according to claim 1 in the preparation of a medicament, wherein the daily dosage of myricitrin is in the range of 12-50mg.

7. The use of myricetin according to claim 1 in the preparation of a medicament, wherein the dosage form of the medicament is one of a tablet, a capsule and a pill.

8. The use of myricetin according to claim 1 in the preparation of a medicament, wherein the medicament is a slow release formulation or a controlled release formulation.

9. The use of myricetin according to claim 1 in the preparation of a medicament, wherein the medicament further comprises at least one of a thrombopoietin receptor agonist, recombinant human thrombopoietin or recombinant human interleukin-11.