CN114366750B - Application of XMU-MP-1 in preparation of medicine for preventing and/or treating immune thrombocytopenia ITP - Google Patents

Abstract

The invention provides an application of XMU-MP-1 in preparation of a medicine for preventing and/or treating immune thrombocytopenia ITP, and belongs to the technical field of medicines. The invention discovers that XMU-MP-1 has the function of promoting platelet recovery after ITP; the effect is realized mainly by that XMU-MP-1 promotes the maturation of megakaryocytes and the expression of surface molecules; and XMU-MP-1 can remarkably promote the mature migration mobility of megakaryocytes, promote the migration movement of the mature megakaryocytes from the bone niche of bone marrow to the blood vessel niche where blood vessels are located and generate enough platelets; this effect is achieved primarily by regulating the cytoskeleton and motility of megakaryocytes.

Description

Application of XMU-MP-1 in preparation of medicine for preventing and/or treating immune thrombocytopenia ITP

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of XMU-MP-1 in preparation of ITP (immune thrombocytopenia) medicines.

Background

Immune Thrombocytopenia (ITP) which is mainly characterized by megakaryocytic maturation disorder is an immune-mediated blood system disease, and the number of platelets is sharply reduced due to increased platelet destruction and myelomegakaryocytic maturation disorder. The clinical manifestations mainly include extensive bleeding of skin mucosa, and life-threatening visceral bleeding may occur, which may result in prolonged illness and serious influence on life quality and even life-threatening. Therapeutic to reduce platelet destruction and promote platelet production, conventional first-line treatment regimens include glucocorticoids, intravenous gamma globulin; second-line treatment regimens include thrombopoietic drugs, rituximab, splenectomy, and other immunosuppressive drugs.

The current clinical treatment regimen for ITP is mainly hormonal therapy, including the glucocorticoid common prednisone; dexamethasone can be used for patients with severe hemorrhage, and prednisone can be taken after symptom relief. The simultaneous administration of large doses of intravenous gamma globulin ensures the pharmaceutical effect. Platelet infusion was selected in the presence of intracranial hemorrhage or acute visceral hemorrhage, with concurrent administration of large doses of adrenocortical hormone to reduce afferent platelet destruction. Due to the particularity of the treatment scheme, a series of adverse reactions are often caused. With the increase of dosage, the use time and the treatment course, the incidence of adverse drug reactions, especially cardiotoxicity, edema and the like is increased, and common adverse reactions comprise: fever, edema, blood pressure increase, cardiovascular system abnormality and anaphylaxis, etc., which seriously affect the prognosis of the patient. Therefore, it is important to find a more effective medicine for treating ITP and a new method for reducing adverse reactions.

The compound XMU-MP-1 has a molecular formula of C ₁₇ H ₁₆ N ₆ O ₃ S ₂ XMU-MP-1, molecular weight 416.48, inhibits Hippo kinase MST1/2 activity and is therefore capable of activating the downstream effector YAP1 protein and promoting cell growth. Treatment with XMU-MP-1 increased the nuclear localization of YAP 1. In a mouse disease model, 1-3mg/kg of XMU-MP-1 is injected into the abdominal cavity of the mouse to the mouse model with acute/chronic liver injury, the XMU-MP-1 has good pharmacokinetics in vivo, and can improve the repair and regeneration of the intestinal tract and the liver of the mouse. XMU-MP-1 can protect mice from DSS-induced colitis and alleviate chronic liver injury.

The prior art for treating ITP and the medicines have more adverse reactions, can cause fever edema, cardiovascular system abnormality and even sudden death and other malignant consequences, and the prior hormone and cytokine medicines can not well radically cure the clinical course of immune thrombocytopenia and can not exert long-acting useful curative effect.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of XMU-MP-1 in preparing a medicine for preventing and/or treating immune thrombocytopenia ITP.

The invention provides an application of XMU-MP-1 or pharmaceutically acceptable salt in preparing a medicament for preventing or/and treating immune thrombocytopenia ITP, wherein the structural formula of XMU-MP-1 is as follows:

in one embodiment of the present invention, the pharmaceutically acceptable salt includes one or more of an inorganic acid salt, an organic acid salt, an alkyl sulfonate salt, and an aryl sulfonate salt.

In one embodiment of the invention, the medicament further comprises a pharmaceutically acceptable carrier.

In one embodiment of the invention, the carrier is selected from one or more of disintegrants, diluents, lubricants, binders, wetting agents, flavouring agents, suspending agents, surfactants and preservatives.

In one embodiment of the invention, the disintegrant is selected from one or more of corn starch, potato starch, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethylcellulose calcium, and alginic acid.

In one embodiment of the invention, the diluent is selected from one or more of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate and crystalline cellulose.

In one embodiment of the invention, the lubricant is selected from one or more of aerosil, magnesium stearate, calcium stearate, stearic acid, talc and anhydrous silica gel.

In one embodiment of the present invention, the binder is selected from one or more of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose, and polyvinylpyrrolidone; the wetting agent is selected from sodium lauryl sulfate; the flavoring agent may be one or more of aspartame, stevioside, sucrose, maltitol and citric acid.

In one embodiment of the invention, the suspending agent is selected from one or more of acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, and aluminum stearate gel; the surfactant is selected from one or more of lecithin, sorbitan monooleate and glyceryl monostearate; the preservative is selected from methyl paraben or/and propyl paraben.

In one embodiment of the present invention, the pharmaceutical dosage form is tablet, capsule, soft capsule, granule, pill, oral liquid, emulsion, dry suspension, dry extract or injection.

The medicine is used for promoting differentiation and maturation of megakaryocyte and platelet production in immune thrombocytopenia. The medicine is used for up-regulating the expression of megakaryocyte differentiation related molecules and megakaryocyte surface characteristic molecules. The medicine is used for promoting megakaryocyte migration and movement and promoting megakaryocyte plate production. The medicine is used for promoting skeleton rearrangement required by megakaryocyte migration movement and platelet production.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the compound XMU-MP-1 is used for evaluating the platelet level in ITP and the effect of megakaryocyte maturation and plate production in the ITP model. Meanwhile, XMU-MP-1 is found to have the function of promoting platelet recovery after ITP; the effect is realized mainly by that XMU-MP-1 promotes the maturation of megakaryocytes and the expression of surface molecules; and XMU-MP-1 can obviously promote the mature migration mobility of the megakaryocyte, promote the migration mobility of the mature megakaryocyte from the bone niche of the bone marrow to the blood vessel niche where the blood vessel is positioned and generate enough platelets; this effect is achieved primarily by regulating the backbone and motility of megakaryocytes.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a graph of the effect of YAP1 of the present invention on megakaryocyte expansion and differentiation maturation. Wherein, A is YAP1 which has no obvious influence on the proliferation of megakaryocytes; the B is YAP1 which has no influence on the formation of megakaryocyte colonies. The up-regulation of C as YAP1 causes the diameter of megakaryocytes to be enlarged; d is XMU-MP-1, which can obviously promote the nuclear entry function of YAP 1. Effect of XMU-MP-1 and Peptide17 on the expression of megakaryocyte surface molecules; total megakaryocyte count (CD 41) when F is upregulated by YAP1 activation ⁺ ) And mature megakaryocytes (CD 41) ⁺ CD42a ⁺ ) The ratio of (A) does not change significantly; g is the effect of XMU-MP-1 and Peptide17 on megakaryocyte plate production. YAP1 up-regulation can promote platelet production, while inhibition of YAP1 activity can significantly reduce platelet production. XMU-MP-1: MST1/2 inhibitors, YAP1 activators; peptide17: inhibitors of YAP1 activity.

FIG. 2 is a graph showing the effect of the YAP1 activators and inhibitors on megakaryocyte adhesion migration plates. Wherein, when A is YAP1 activation expression up-regulation, megakaryocyte production plates are obviously increased; after the YAP1 activity is inhibited, the yield of the plate is obviously reduced; B-C is YAP1 for promoting megakaryocyte adhesion and spreading; D-E is YAP1 ability to promote megakaryocyte migration; XMU-MP-1: MST1/2 inhibitors, YAP1 activators; peptide17: inhibitors of YAP1 activity.

FIG. 3 is the molecular mechanism of the activators and inhibitors of YAP1 of the present invention for plating megakaryocyte migration. Wherein, A is the change of the skeleton of megakaryocyte and the formation of platelet, YAP1 activation obviously promotes the rearrangement of the cell skeleton; b is YAP1 activation to enhance the formation of megakaryocyte internal stress fibers; C-D is the expression condition of a cytoskeleton related protein Merlin in megakaryocytes, wherein the Merlin is regulated by YAP1 and negatively regulates the rearrangement of cytoskeletons and the formation of stress fibers; e is the case of the megakaryocyte signaling pathway. XMU-MP-1: MST1/2 inhibitors, YAP1 activators; peptide17: inhibitors of YAP1 activity.

FIG. 4 is the molecular mechanism of YAP1 in the regulation of the DAMI skeleton of the megakaryocyte cell line. Wherein, A is the identification of the overexpression and the knockdown effect of YAP1 in the DAMI of the megakaryocyte line; b is the effect of YAP1 on the adhesive capacity of DAMI cells; c is the effect of YAP1 on the migratory capacity of DAMI cells. D is the effect of YAP1 on the DAMI cytoskeleton; E-F is the effect of YAP1 on the activation of megakaryocyte signaling pathway and the expression of cytoskeleton-associated protein Merlin.

FIG. 5 is a graph showing the effect of XMU-MP-1 of the present invention on the regulation of mouse ITP. Wherein A is the model construction and administration scheme of ITP; b is the recovery condition of the platelets after the ITP model is treated by XMU-MP-1 and Vertporfin; the effect of C-E being XMU-MP-1 and Vertporfin on the number and distribution of megakaryocytes in bone marrow sections.

FIG. 6 shows XMU-MP-1 vs. YAP1 of the present invention ^+/- Regulation of mouse ITP. Wherein A is the model construction and administration scheme of ITP; b is XMU-MP-1 to WT and YAP1 ^+/- Recovery of platelets after ITP model; C-E is the influence of XMU-MP-1 on the number and distribution of megakaryocytes in the bone marrow slices respectively; f is the effect of XMU-MP-1 on the regulation of megakaryocytoskeleton in bone marrow.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Examples

A, materials and methods

(I) animals

6-8 week SPF grade C57BL/6J (WT) mice, YAP1, purchased from the Experimental animals center of Suzhou university ^+/- The mice were purchased from Jiangsu Jiejiaokang Biotechnology GmbH and were raised in a university Standard animal center maintaining an SPF-level environment. All experimental mice are kept in a constant environment, the raising environment requires the temperature of 18-29 ℃, the daily temperature difference is less than or equal to 3 ℃, the relative humidity reaches 40-70 percent, the fresh air ventilation frequency is l0 times/h, the airflow speed is less than or equal to 0.18m/s, the pressure difference is 25Pa, the cleanliness is ten thousand grades, and the ammonia concentration is l5mg/m ³ Noise less than or equal to 60dB, illumination intensity of 150-300 Lux, illumination period (12 hours of illumination: 12 hours of darkness), all mice are raised in isolation cages at SPF (specific pathogen-free) level, and pathogenic bacteria are not existedAnd sufficient water and food are ensured. All procedures were performed according to the guidelines for the care and use of animals by the animal care committee and the national institutes of health.

(II) cells

Primary cells: separation of human peripheral blood sample to obtain primary CD34 ⁺ Hematopoietic stem cell separation induces megakaryocyte differentiation and maturation. The megakaryocyte differentiation induction medium is composed of SFEM medium containing TPO (100 ng/ml), SCF (100 ng/ml) and IL-3 (10 ng/ml). Half a weekly change of medium during the cultivation, 37 ℃ C., 5% CO ₂ After 12 days of induction, mature megakaryocytes can be obtained. Primary CD34 was subsequently treated with an activator of YAP1 (XMU-MP-1) ⁺ Hematopoietic Stem Cells (HSCs) were observed for the role and effect of the maturation process.

Megakaryocyte line DAMI: human megakaryocyte cell line DAMI was cultured in RPMI1640 complete medium containing 10% FBS, 37 ℃,5% ₂ Culturing in a cell culture box. YAP1 overexpression and interference plasmids were transferred into DAMI cell lines by lentiviral vectors to observe the effect of YAP1 protein on megakaryocytes.

(III) main reagent and consumable

XMU-MP-1

Vertporfin MCE Inc., china

CD41a/CD42a/CD61 BD company, USA

CD34 kit: STEM CELL, USA

RPMI1640 medium: hyclone, china

Fetal bovine serum: BI Ltd, china

Penicillin streptomycin: solebao, china

Hematoxylin eosin staining kit: solebao, china

CD41/YAP 1/CFL/ACTIN/antibody: CST, USA

RIPA lysate (Strong), biyunshi Biotechnology, china

10 Xelectrophoresis buffer (pH8.5): 30.2g of Tris base; 188g of glycine; 10% SDS 100mL; adding 900mL of triple distilled water for dissolution, adjusting the pH to 8.5 by hydrochloric acid, and then carrying out constant volume to 1L.

10 × wet transfer buffer: 30.3g of Tris base; 144g of glycine; adding 1L of triple distilled water for dissolving and fixing the volume.

1 × wet transfer buffer: 100mL 10 × wet buffer; 200mL of anhydrous methanol solution; adding triple distilled water to a constant volume of 1L.

10 × TBS: tris base 22.4g; 80g of NaCl and hydrochloric acid are used for adjusting the pH value to 7.6, and then triple distilled water is added to the volume of 1L.

1 × TBS-T: to 1L of 1 × TBS, 1mL of Tween-20 (i.e., 0.1%) was added.

Western sealing fluid: 5% skimmed milk (prepared with 1 × TBS, preferably ready to use).

PageR μ lerPrestatinedProtein Ladder (cat # 26617, thermo Fisher, USA).

Phosphatase/Protease inhibitor Cocktail (cat. No. 5872, cell Signaling Technology, U.S.A.).

(IV) Experimental methods

1. Differentiation induction culture of human megakaryocytes

Obtaining peripheral blood cells mobilized by GM-CSF, CD34 ⁺ Hematopoietic stem cells were sorted using a magnetic sorting kit and cultured in SFEM medium consisting of TPO (100 ng/mL), SCF (100 ng/mL), and IL-3 (10 ng/mL) for 12 days. Half a weekly change of medium during the cultivation, 37 ℃ C., 5% CO ₂ After 12 days of induction, mature megakaryocytes can be obtained.

2. DAMI culture of megakaryocyte line

Human megakaryocyte cell line DAMI was cultured in RPMI1640 complete medium containing 10% FBS, 37 ℃,5% ₂ Culturing in a cell culture box. YAP1 overexpression and interference plasmids were transferred into DAMI cell lines by lentiviral vectors to observe the effect of YAP1 protein on megakaryocytes.

3. Cell surface flow antibody staining

After collecting megakaryocytes, the cells were fixed in 4% PFA, washed with PBS, and stained with a flow antibody such as anti-human CD41/CD42/CD61 at 37 ℃ for 30 minutes at room temperature. After PBS washing, the proportion and population of cells expressing antibody positivity were analyzed by flow cytometry.

4. Cellular immunofluorescence staining

The cells were fixed in 4-percent PFA for 10 minutes, permeabilized with 0.1-percent Triton X-100, followed by immunostaining for 1 hour at room temperature and incubation with primary antibody overnight at 4 ℃ after blocking. At the end of the primary antibody incubation, the cells were washed with PBS and specific proteins were visualized using fluorescently labeled secondary antibody. Finally nuclei were stained with DAPI. And (4) sealing the anti-quenching sealing agent. The staining results were observed using a laser confocal microscope.

5. Megakaryocyte producing plate

Megakaryocytes matured for 12 days in induced culture were collected, and were placed on a fibrinogen (Fg, 200 μ g/mL) coated surface, after 5 hours, megakaryocytes adhered to the surface and produced Pseudopodiform Platelets (PPF), from which the platelet-producing function of megakaryocytes was investigated.

6. Adhesion test of megakaryocytes

Mature megakaryocytes and megakaryocyte cell lines were collected, the megakaryocytes were placed on the surface coated with fibrinogen Fg, and the number of megakaryocytes adhering to the surface of Fg was measured after 3 hours, whereby the adhesion ability of the megakaryocytes was evaluated.

7. Megakaryocyte migration assay

Collecting mature megakaryocytes and megakaryocyte cell lines, placing the megakaryocytes in SFEM culture medium without cell factors into an upper chamber of a migration chamber with the aperture of 8 mu m, adding SFEM culture medium with double TPO concentration into a lower chamber, inducing the megakaryocytes to differentiate, detecting the number of the megakaryocytes migrating to the lower layer of the migration chamber after 24 hours, and judging the migration capacity of the megakaryocytes according to the detection result.

8. Immunoblotting experiment

Megakaryocytes were collected, lysed on ice for 30 minutes using RIPA protein lysate containing protease inhibitors and phosphatase inhibitors, and the DNA was disrupted by sonication, which changed the original viscous state to clear. Adding 5 × loading buffer, boiling at 100 deg.C for 10 min, quickly taking out, cooling on ice, and directly storing at-20 deg.C.

Preparing SDS-PAGE gel with proper concentration according to the size of target protein, loading 20 mu g of the SDS-PAGE gel for electrophoresis, then transferring the SDS gel to a PVDF membrane by 200mA, transferring the protein to the PVDF membrane, adding primary antibody after sealing operation, shaking and incubating overnight at 4 ℃, washing off non-specific primary antibody coloring by TBST, then shaking and incubating HRP-labeled secondary antibody for 1 hour at room temperature, and developing after washing to determine the activation and expression level of the protein.

9.RNA extraction

Megakaryocytes were collected, added to 600. Mu.L RNAiso Plus/tube, lysed thoroughly, allowed to stand on ice for 5 minutes, centrifuged at 12000rpm for 5 minutes at 4 ℃ and the supernatant was transferred to a new EP tube. Adding 100 μ L chloroform, covering, shaking vigorously for 15s, separating phases, standing at room temperature for 5 min, centrifuging at 12000rpm for 15 min at 4 deg.C, separating three layers, transferring supernatant into new EP tube, and allowing the supernatant to absorb into white middle layer. Add 300 u l isopropanol, reverse the top and bottom fully mixing, room temperature static 10 minutes, 4 degrees C, 12000rpm 10 minutes centrifugal, remove the supernatant, can see the EP tube bottom has RNA precipitation. Add 1mL 75% ethanol (gun head does not touch the sediment), gently upside down wash the tube wall, centrifuge at 12000rpm for 5 minutes at 4 deg.C, remove the supernatant, dry the tube wall ethanol filter paper, dry the sediment at room temperature for 5 minutes, add 30. Mu.L DEPC water to dissolve the RNA sediment, fully dissolve, store at-80 deg.C.

10. Quantitative fluorescence QPCR

Reverse transcription of RNA: the reaction system (20 uL/tube) contains 1000ngRNA and 5 Xreverse transcription Mix Buffer4 uL, sterile water is added until the total volume is 20 uL, centrifugation is carried out after mixing, reverse transcription is carried out by a PCR instrument, and the reverse transcription conditions are as follows: the samples were stored at 37 ℃ for 15 minutes, 85 ℃ for 5 seconds and 4 ℃.

Real-time PCR detection of target gene mRNA expression level: designing primers aiming at a target gene and a housekeeping gene GAPDH; then prepare Real-time PCR reaction system (96-well plate, 20. Mu.L/tube)

20 μ L system:

2×SYBER green 10μL

primer F (20. Mu.M) 0.5. Mu.L

Primer R (20. Mu.M) 0.5. Mu.L of LDEPC water 8. Mu.L

cDNA template (reverse transcription product) 1. Mu.L

The Real-time PCR system in each tube was added to a specific 96-well plate and placed in a Real-time quantitative PCR instrument for amplification. Statistical method of results: the housekeeping gene GAPDH is used as an internal reference, the expression of the housekeeping gene GAPDH in tissues is stable, and a delta Ct internal reference method is adopted in obtained data.

a. Reference gene normalization sample differences: ct target gene-Ct reference gene = Δ Ct.

b. And (3) sample comparison: Δ Ct experimental samples- Δ Ct control samples = Δ Δ Ct.

c. Using the formula to calculate: fold change =2 ^-ΔΔCt To calculate the change of the sample in the experimental group relative to the sample multiple of the control group.

YAP1 overexpression and interfering cell line construction

Lentiviral vectors overexpressing human YAP1 and shYAP1 were prepared using the VSV-G lentivirus expression packaging system and lentivirus replication was performed by co-transfecting a mixture of pCDH-hYAP1/pCDH/Plko.1-ShYAP1/Plko.1 plasmid and three packaging plasmids (. DELTA.R, rev and VSVG) into HEK-293T cells. Viral supernatants collected after 48 or 72 hours were filtered through a 0.45 μm filter and stored at-80 ℃. Viral supernatants from different groups were subsequently added to the megakaryocyte cell line DAMI at a multiplicity of infection (MOI) of 50. Thereby constructing YAP1 over-expression cell lines and YAP1 expression interference cell lines.

12. Mouse ITP model construction

Adult WT mice weighing 20-24g at 6-8 weeks were administered Integrin alpha IIb (MWReg 30) at a dose controlled at 200. Mu.g/kg, 20. Mu.L of antibody was taken, then supplemented to 100. Mu.L with 1xPBS, prepared as an antibody solution, and then administered by intraperitoneal injection once a day for 7 days, to the completion of the construction of the ITP mouse model, followed by observation of platelet recovery in ITP mice and megakaryocyte maturation and distribution in mouse bone marrow.

ITP histopathological examination

Constructing an ITP model by the mice, and observing platelet recovery conditions of mice of different treatment groups through different administration schemes; after the hemogram of the mouse returns to a normal level, the mouse is killed and the femoral neck bone of the mouse is obtained by separation, after a series of operations such as fixation, decalcification, embedding and the like, the femoral bone of the mouse is frozen and sliced, then the immunofluorescence of the marrow slice is carried out, and the maturation of megakaryocytes in the marrow of the mouse and the distribution condition of the megakaryocytes are observed.

14. Mouse bone marrow section staining

The femurs of the mice were cryosectioned at a thickness of 8 μm and subsequently fixed with methanol using immunohistochemical blocking solutions, and also blocked with goat serum working solutions. Blocking was performed followed by incubation of sections with primary antibody or isotype IgG controls overnight at 4 ℃, followed by fluorescent-labeled secondary antibody for 30 min, finally staining of the nuclei with the nuclear dye DAPI, and finally blocking with anti-quenching blocking tablets.

15. Statistical analysis

Statistical analysis was performed using Prism 8.0 (GraphPad) software. Comparisons of continuous variables from different groups were performed using unpaired Student's t test (two-tailed), mann-whitney u test, one-way or two-way ANOVA test, and post hoc analysis. Chi-square test is used to compare categorical variables. A two-tailed P value <0.05 was considered to be a statistically significant difference.

Second, experimental results

The regulating effect of an activator XMU-MP-1 and an inhibitor Peptide17 of a Hippo signal channel core element YAP1 on the differentiation and maturation of megakaryocytes.

Separating to obtain primary CD34 ⁺ After hematopoietic stem cells, megakaryocyte-inducing differentiation medium was induced and then on day 0, a defined concentration of activator XMU-MP-1 (300 nM) and inhibitor Peptide17 (2. Mu.M) of YAP1 was added twice a week half-way replacement, 37 ℃,5% CO ₂ After 12 days of induction, mature megakaryocytes can be obtained.

First, the present inventors examined the proliferation of cells and the formation of megakaryocyte colonies during the culture process, and found that YAP1 had no significant effect on the amplification of megakaryocytes (FIG. 1A) and the formation of megakaryocyte colonies (FIG. 1B). Subsequently, in the culture process, the invention discovers that from day 5, the activating agent XMU-MP-1 can remarkably promote the morphological development of the megakaryocytes, and compared with the control group and the inhibitor Peptide17 treatment group, the volume of the megakaryocytes of the XMU-MP-1 group is remarkably increased, and the specific gravity of the megakaryocytes with large volume is remarkably increased (FIG. 1C). The activating agent XMU-MP-1 is suggested to obviously promote the morphological development of megakaryocytes and the volume size of the megakaryocytes. Research results show that after the activity of the Hippo signal channel is inhibited, the morphological development of megakaryocytes can be obviously promoted when the expression of the nucleus-entering YAP1 is up-regulated.

Along with the extension of the culture time, the invention discovers that YAP1 in megakaryocytes has obvious nuclear entry phenomenon after being induced by YAP1 activator XMU-MP-1 for 12 days, while the YAP1 expression of an inhibitor Peptide17 treatment group is obviously reduced and does not enter the nucleus to activate the transcription of downstream initiation genes. Subsequently, megakaryocytes are collected, the expression condition of the gene related to the megakaryocytes is detected by a QPCR mode, and the result shows that the activator XMU-MP-1 can obviously promote the expression of the CD41a and the gene CD42 related to the maturation of the megakaryocytes (as shown in figure 1E), and then the megakaryocyte antibody CD41a and the mature megakaryocyte surface antibody CD42a are marked in a flow mode. The results show that the specific gravity of the total megakaryocytes (CD 41 a) after treatment with XMU-MP-1, the activator of YAP1, and the Peptide17, the inhibitor of YAP1 activity ⁺ ) And the specific gravity of mature megakaryocytes in megakaryocytes (CD 41 a) ⁺ CD42a ⁺ ) And not significantly changed (see fig. 1F). However, as a result of examining CD61, which is an important platelet-producing molecule of megakaryocytes, and the production ratio of platelets, it was found that XMU-MP-1, which is an activator, can significantly promote the expression level of CD61 and the production ratio of platelets. And the phenomenon is obviously reduced after being treated by inhibitor Peptide 17. It is suggested that YAP1 has no obvious change in the specific gravity of the total megakaryocytes and the mature megakaryocytes, but has obvious regulation and control effect on the mature plate-producing process of the megakaryocytes (as shown in FIG. 1G).

Effect of xmu-MP-1 and inhibitor Peptide17 on plating of megakaryocyte adhesion migration.

Through the previous research, the invention discovers that XMU-MP-1 can obviously promote the morphological development of megakaryocytes and the plate production ratio of the megakaryocytes in the culture process. To further verify the previous experimental data, the present invention collected megakaryocytes matured by induction culture for 12 days, placed the megakaryocytes on the surface coated with fibrinogen (Fg, 200 μ g/mL), and within 5 hours, the megakaryocytes adhered to the surface and generated pseudo-Podiform Platelets (PPF), thereby exploring the platelet production function of the megakaryocytes. The results showed that the XMU-MP-1 treated group had significantly increased PPF in megakaryocytes compared to the normal non-treated group, while the inhibitor Peptide17 treated group had a sharply decreased production of PPF. The XMU-MP-1 can obviously promote the mature of the megakaryocyte to produce the plate; the inhibition of YAP1 in megakaryocytes will significantly decrease the plate-producing rate of megakaryocytes (FIG. 2A).

The present inventors have found that, in the differentiation and maturation of megakaryocytes, the migration and adhesion ability of megakaryocytes is closely indistinguishable from the megakaryocytes, and the plating ratio of megakaryocytes is significantly changed, and thus, whether or not the migration and adhesion ability of megakaryocytes is also changed to a different degree? Therefore, the invention detects the adhesion capability and the migration capability of the megakaryocytes through an adhesion experiment and a Transwell migration chamber experiment, and research results show that when XMU-MP-1 promotes the activation expression of YAP1 to be up-regulated, the adhesion and spreading ratio (shown as figure 2B.C) and the migration capability (shown as figure 2D.E) of the megakaryocytes are obviously enhanced compared with a non-treatment group; when the YAP1 activity is inhibited, the adhesion ability and the migration ability of the megakaryocytes are obviously hindered. It is suggested that XMU-MP-1 regulates the process of megakaryocyte plating or is achieved by regulating the migratory adhesion ability of megakaryocytes.

The molecular regulation mechanism of XMU-MP-1 on megakaryocyte maturation and plate production.

Under the physiological state of cells, cytoskeleton plays an important role not only in maintaining cell morphology, bearing external forces, maintaining the order of the internal structure of cells, but also in many important vital activities. The process of megakaryocyte plating is inseparable from the change of cytoskeleton, and the change of cytoskeleton structure directly causes megakaryocyte migration and movement from the generated and matured bone niche to the blood sinus where the vein blood vessel is located, and then the change of the cytoskeleton causes the further extension of the pre-platelet and the relocation of organelles (such as mitochondria) in the pre-platelet (proplastlet). While cytoskeletal changes are also involved in the eventual shedding and division of platelet formation in the bone marrow sinusoidal blood flow. Throughout this process, actin F-actin undergoes continuous remodeling during pre-thrombopoiesis and serves as a motor for megakaryocyte migration in the bone marrow.

In order to further verify the experimental data in the previous stage, the invention collects the megakaryocytes matured after 12 days of induced culture, and observes the distribution of megakaryocyte surface protein and skeleton (Tubulin/Actin) by immunofluorescence staining. As a result of research, the skeletal microtubule (Tubulin) and microfilament (Actin) distribution of megakaryocytes in the XMU-MP-1 group is obviously changed compared with the control group, and the skeletal Actin F-Actin obviously activates rearrangement and forms Stress fibers (Stress fiber). The suggestion that XMU-MP-1 can obviously promote the movement migration capability of megakaryocytes is realized by regulating and controlling the skeletal rearrangement and the formation of stress fibers (as shown in figure 3 A.B).

Subsequently, in order to further explore the molecular mechanism of YAP1 in modulating cytoskeleton and related regulatory molecules, the present invention analyzed the expression of Merlin (also called NF 2) that is indivisible to maintaining cytoskeleton stability. Merlin is known to bind and stabilize microtubules by reducing the rate of microtubule polymerization and depolymerization and reducing the frequency of microtubule catastrophes, by attenuating tubulin turnover. Meanwhile, merlin maintains the stability of cytoskeleton by binding to actin and binding to membrane proteins, i.e., merlin, an important binding protein of cytoskeleton, stabilizes the structure of cytoskeleton, acting as a link between cytoskeletal components and cell membranes. The invention detects the expression and activation conditions of Merlin in megakaryocytes of the XMU-MP-1 group and the active inhibitor Peptide17 group, and the result shows that, consistent with the expectation, compared with the normal condition, the XMU-MP-1 can cause the expression of Merlin to be obviously reduced, thereby promoting the rearrangement of cytoskeleton and causing the migration movement of megakaryocytes or the formation of proplatelets to be obviously enhanced; when YAP1 activity was inhibited, merlin expression was significantly up-regulated, which in turn retarded cytoskeletal rearrangement and stress fiber formation, significantly hampered cell motility and plate-producing ability (see fig. 3 c.d).

The migration adhesion and the skeleton rearrangement of the cells have an inseparable relationship with the activation of the plate production and MAPK signal channels, and in order to further verify the activation condition of the cytoskeleton signal channels related to the process of transferring the plate production, the invention collects the megakaryocytes mature by induced culture for 12 days, and detects the activation condition of the signal channels after cracking proteins. The result shows that the activation of XMU-MP-1 can lead to the significant reduction of the activation of ERK1/2 and Cofilin (CFL), thereby promoting the rearrangement of cytoskeleton and the migration movement of cells, and leading to the significant enhancement of the formation of megakaryocyte pre-platelets; when the activity of YAP1 is inhibited, the activation of ERK1/2 and CFL is significantly up-regulated, and the rearrangement of cytoskeleton and the formation of stress fibers are blocked (see FIG. 3E).

The regulation and molecular mechanism of YAP1 on the cytoskeleton of the megakaryocyte cell line DAMI.

Through the induction and culture of the primary megakaryocytes, the research of the invention discovers that XMU-MP-1 participates in the regulation of the maturation and plate production process of the megakaryocytes, and the regulation of the maturation and plate production of the megakaryocytes is realized through the regulation of the change of cytoskeletons and the activation of a framework-related signal channel. In order to further verify the result of the primary cells and the regulation and control effect of YAP1, the invention constructs an overexpression cell line (PCDH-YAP 1-DAMI) of YAP1 and a cell line (Plko.1-YAP 1-sh-DAMI) with reduced interference in a megakaryocyte cell line, and further observes the regulation and control effect of YAP1 on the DAMI of the megakaryocyte.

The invention detects the adhesive migration and skeleton rearrangement abilities of YAP1 to megakaryocytes through cell adhesion, cell migration, immunofluorescence and immunoblotting experiments respectively. The results of the study found that, in line with the results of primary cells, YAP1 overexpression significantly enhanced megakaryocyte adhesion and migration, cytoskeletal rearrangement, and stress fiber formation. And the process is realized by the expression inhibition of cytoskeleton related protein Merlin and the activity inhibition of ERK/CFL. When YAP1 is knocked down, activation of ERK and CFL is up-regulated, so that rearrangement of cytoskeleton and formation of stress fibers are blocked, and adhesion migration of megakaryocytes is inhibited (as shown in FIG. 4).

Effect of YAP1 agonist XMU-MP-1 on distribution and number of platelets, megakaryocytes, megakaryocyte maturation and plating process in the hematopoietic System in mouse ITP model

And (3) constructing an ITP model of the WT mouse, and verifying the effect of the XMU-MP-1 in the process of megakaryocyte differentiation maturation and plate production through an animal model. ITP mice were constructed and subsequently intervened with XMU-MP-1 therapy while observing the effect of different treatments on mouse hemogram recovery and the development and maturation of hematopoietic megakaryocytes and platelet production and function using the YAP1 activity inhibitor, verteporfin. The result proves that the YAP1 agonist compound XMU-MP-1 can obviously promote the recovery of platelet level after ITP and the differentiation and maturation of megakaryocytes in bone marrow, and the distribution condition of the megakaryocytes in the bone marrow is also obviously improved, so that the molecular mechanism of the compound XMU-MP-1 for regulating and controlling the megakaryocyte maturation production plate is verified, the compound XMU-MP-1 is expected to diagnose and treat ITP, and important experimental basis and theoretical basis are provided for the clinical treatment and diagnosis of the megakaryocyte maturation disorder hematological diseases.

YAP1 agonist XMU-MP-1 vs. YAP1 ^+/- Effect of platelet, megakaryocyte distribution and number, megakaryocyte maturation, and plating Process in the hematopoietic System in the mouse ITP model

Construction of WT and YAP1 ^+/- The mouse ITP model verifies the function of YAP1 agonist XMU-MP-1 in the process of megakaryocyte differentiation maturation and plate production in the process of ITP pathogenesis through an animal model. ITP mice were constructed and subsequently intervened in ITP mice using XMU-MP-1 therapy, observing the effect of restoring YAP1 levels to some extent by XMU-MP-1 and thus on mouse hemogram restoration and the development and maturation of hematopoietic megakaryocytes and platelet production and function. The results confirm that YAP1 agonist compound XMU-MP-1 can obviously recover YAP1 ^+/- The platelet level is restored after ITP caused by YAP1 deficiency, megakaryocytes in bone marrow are differentiated and matured, and the distribution condition of the megakaryocytes in the bone marrow is also obviously improved, so that the molecular mechanism of the compound XMU-MP-1 for regulating and controlling the megakaryocyte maturation production plate is directly verified, the compound XMU-MP-1 is prompted to be expected to diagnose and treat ITP, and important experimental basis and theoretical basis are provided for clinical treatment and diagnosis of the megakaryocyte maturation disorder hematological diseases.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims (12)

1. The application of XMU-MP-1 or pharmaceutically acceptable salt in the preparation of a medicament for preventing and/or treating immune thrombocytopenia ITP, wherein the structural formula of XMU-MP-1 is as follows:

   

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2. 2. use according to claim 1, characterized in that the pharmaceutically acceptable salt is selected from inorganic and/or organic acid salts.
3. 3. The use according to claim 2, wherein the organic acid salt is selected from one or both of alkyl sulfonate and aryl sulfonate.
4. 4. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.
5. 5. The use according to claim 4, wherein the carrier is selected from one or more of disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants and preservatives.
6. 6. Use according to claim 5, wherein the disintegrant is selected from one or more of corn starch, potato starch, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethylcellulose calcium and alginic acid.
7. 7. Use according to claim 5, wherein the diluent is selected from one or more of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate and crystalline cellulose.
8. 8. Use according to claim 5, wherein the lubricant is selected from one or more of aerosil, magnesium stearate, calcium stearate, stearic acid, talc and anhydrous silica.
9. 9. The use according to claim 5, wherein the binding agent is selected from one or more of acacia, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose and polyvinylpyrrolidone; the wetting agent is selected from sodium lauryl sulfate; the correctant is one or more of aspartame, stevioside, sucrose, maltitol and citric acid.
10. 10. Use according to claim 5, wherein the suspending agent is selected from one or more of acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose and aluminium stearate gel; the surfactant is selected from one or more of lecithin, sorbitan monooleate and glyceryl monostearate; the preservative is selected from methyl paraben or/and propyl paraben.
11. 11. The use according to claim 1, wherein the medicament is in the form of tablets, capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts or injections.
12. 12. The use according to claim 1, wherein the medicament is in the form of a soft capsule.

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