Use of mycophenolate mofetil in the preparation of an antithrombotic medicament

Abstract

The application provides application of mycophenolic acid methyl ester in preparation of an anti-thrombus drug and belongs to the technical field of anti-thrombus drugs. The application first discovers the anti-thrombus activity of the compound mycophenolic acid methyl ester, and provides experimental support and theoretical basis for development of a novel drug for treating or preventing thrombus.

Description

Technical Field

[0001] This invention belongs to the field of antithrombotic drug technology, specifically relating to the application of mycophenolate methyl ester in the preparation of antithrombotic drugs. Background Technology

[0002] Thrombosis, as a complex pathological process, seriously threatens human health and is closely related to the occurrence and development of various major diseases such as myocardial infarction, ischemic stroke, and venous thromboembolism. In recent years, the global incidence of thrombosis-related diseases has been on a continuous upward trend, significantly increasing the risk of disability and death for patients and imposing a heavy economic burden on public health systems and families. Existing antithrombotic drugs have significant limitations: a narrow therapeutic window makes it difficult to balance the risk of bleeding; complex interactions with many commonly used drugs may reduce efficacy or cause adverse reactions; long-term use may also lead to safety issues such as gastrointestinal mucosal damage and bone metabolic disorders. Therefore, there is an urgent need to develop new antithrombotic drugs.

[0003] Zebrafish, as an emerging model organism, exhibit unique advantages in the field of thrombosis research. Their short reproductive cycle and large egg production per spawning cycle allow for the rapid acquisition of a large number of experimental samples, significantly improving the efficiency of screening for active compounds. Furthermore, the zebrafish genome shares over 70% homology with the human genome, and the molecular mechanisms and pathological processes of thrombosis are highly similar to those in humans. Active compounds screened using zebrafish models have higher clinical translational potential, providing an efficient and reliable research tool for the development of novel antithrombotic drugs.

[0004] In the field of constructing zebrafish thrombosis models, various modeling methods have been developed using existing technologies. Currently commonly used zebrafish thrombosis inducers include arachidonic acid (AA), ferric chloride, ponatinib, and phenylhydrazine. Ferric chloride and phenylhydrazine initiate coagulation mechanisms by oxidatively damaging the vascular endothelium, while ponatinib activates platelets by inducing vascular endothelial dysfunction. However, these methods either differ from the physiological thrombosis formation mechanism in humans or suffer from operational complexity and poor stability. In contrast, arachidonic acid, through metabolism, produces thromboxane A2 (TXA2), which specifically activates platelets, promotes platelet adhesion, aggregation, and vasoconstriction, and can rapidly induce platelet thrombosis. This gives it a unique advantage in simulating the physiological thrombosis formation mechanism in humans, and thus it has attracted widespread attention.

[0005] However, using arachidonic acid (ARA) as a zebrafish thrombosis modeling reagent still has significant limitations. As a highly active polyunsaturated fatty acid, ARA is easily inactivated by oxidation, light, and other factors. Therefore, high-purity (≥98% HPLC grade) products must be used, and strict light-protection and low-temperature storage measures must be taken. Currently, the market price of research-grade ARA is high, with a reference standard of ≥98% purity costing as much as 6300 yuan / gram, significantly increasing experimental costs. More importantly, existing conventional modeling methods suffer from low thrombosis formation efficiency, typically requiring the ARA concentration to be increased to 80-100 μM and the treatment time extended to more than 1 hour to achieve acceptable modeling results. This high-concentration, long-duration treatment method not only significantly increases reagent consumption but also significantly prolongs the experimental cycle, leading to a double increase in reagent and time costs, severely restricting high-throughput antithrombotic drug screening research based on zebrafish models. Therefore, developing a low-cost, efficient, and stable zebrafish thrombosis model has become a key technical problem that urgently needs to be solved in the current field of antithrombotic drug development. There is currently no information on constructing stable genetic structures. CALR del52 Reports on overexpression zebrafish models.

[0006] The compound mycophenolic acid methyl ester (MAE) is a commercially available product, CAS number 31858-66-9, with the following structural formula: Figure 11 As shown. Currently, there are no reports on the antithrombotic activity of mycophenolate methyl ester. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides the application of mycophenolate methyl ester in the preparation of antithrombotic drugs.

[0008] This study used a zebrafish model of thrombocytosis with thrombosis to screen compounds for antithrombotic activity and found that methyl mycophenolate mofetil has an antithrombotic effect.

[0009] The technical solution of the present invention is as follows: Application of mycophenolate methyl ester in the preparation of antithrombotic drugs.

[0010] According to a preferred embodiment of the invention, the drug contains one or more pharmaceutically acceptable carriers or excipients.

[0011] More preferably, the excipient is at least one of a sustained-release agent, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, or lubricant.

[0012] According to a preferred embodiment of the present invention, the dosage form of the drug is a capsule, pill, tablet, oral liquid, granule, tincture, or injection.

[0013] According to a preferred embodiment of the present invention, the antithrombotic drug includes drugs for treating myocardial infarction, ischemic stroke, or deep vein thrombosis of the lower extremities.

[0014] The beneficial effects of the present invention include at least the following: 1. This invention is the first to discover through experiments that the compound mycophenolic acid methyl ester can reduce the formation of thrombi in blood vessels, inhibit the expression of thrombosis-related factors such as TXA2, vWF, and D-dimer, and improve thrombosis-related pathological processes, thus exhibiting good antithrombotic effects and providing a new and effective candidate substance for the prevention and treatment of thrombotic diseases.

[0015] 2. Methyl mycophenolate mofetil can be used to prepare antithrombotic drugs, which can be used to prevent or treat thromboembolic diseases such as myocardial infarction, ischemic stroke, and deep vein thrombosis of the lower extremities. Attached Figure Description

[0016] Figure 1 For Tol2-EF1a-h CALR del52 -IRES2-mcherry-UTRsv40 recombinant expression vector map.

[0017] Figure 2 For F1 generation CALR del52 Overexpression zebrafish model construction diagram; In the diagram: A represents... CALR del52 Fluorescence microscopy of overexpressing zebrafish; B is electrophoresis of PCR amplification products; the first lane on the left is the 2000 bp marker: from bottom to top, 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 1500 bp, 2000 bp; the second lane is the F1 generation genome; the third lane is the template-free negative control; C is a representative peak diagram of the F1 generation sequencing results; the left box shows a partial EF1a promoter sequence, and the right box shows the CALR sequence. del52 Partial sequence.

[0018] Figure 3 For F2 generation CALR del52 Representative peak diagram of sequencing results from overexpression zebrafish model; In the diagram: the left box shows a portion of the EF1a promoter sequence, and the right box shows the CALR sequence. del52 Partial sequence.

[0019] Figure 4 A statistical chart of the number of circulating platelets in zebrafish; compared with the same group Tg(cd41:eGFP) compared to, ** P <0.01.

[0020] Figure 5 A statistical graph showing the fluorescent area and fluorescence intensity of platelets in the tail of zebrafish. In the figure: A shows platelet aggregation in the zebrafish tail; B shows the statistical graph of fluorescent area of ​​platelets in the tail; C shows the statistical graph of fluorescent intensity of platelets in the tail. (Compared with the same group) Tg(cd41:eGFP) compared to, ** P <0.01.

[0021] Figure 6 A statistical graph of blood flow velocity in the caudal artery of a zebrafish; With the same group Tg(cd41:eGFP) compared to, ** P <0.01, *** P <0.001.

[0022] Figure 7 for Tg (cd41: eGFP; EF1a: CALR del52 ） Statistical chart of thrombosis-related factors in transgenic zebrafish; In the chart: A represents thromboxane A2 (TXA2) content, B represents von Willebrand factor (vWF) content, C represents tissue factor (TF) content, and D represents D-dimer content; compared with the same group Tg(cd41:eGFP) compared to, ** P <0.01, *** P <0.001.

[0023] Figure 8 Statistical graph of caudal artery flow velocity in zebrafish treated with different concentrations of arachidonic acid for different durations; In the figure: A is a statistical graph of blood flow velocity in the tail artery; B is a representative graph of the blood vessels in the tail of zebrafish treated with 20 μM arachidonic acid for 20 min; the red dashed box indicates the site of thrombus formation; and... Tg(cd41:eGFP) Compared to the control group, # P <0.05, ## P <0.01, ### P <0.001; AA represents arachidonic acid.

[0024] Figure 9 Statistical graphs of circulating platelet count, tail platelet aggregation area, and fluorescence intensity in zebrafish; In the figure: A is a representative image of platelet aggregation in the tail of zebrafish from different treatment groups; B is the number of circulating platelets; C is the area of ​​platelet aggregation in the tail; and D is the fluorescence intensity of platelets in the tail. Compared with the cd41 group, &P <0.05, &&& P <0.001; with CALR del52 Compared to the group, # P <0.05, ## P <0.01; compared with CALR del52 Compared to group +AA ** P <0.01, *** P <0.001; AA represents arachidonic acid, and ASA represents aspirin.

[0025] Figure 10 A statistical graph of blood flow velocity in the caudal artery of a zebrafish; In the figure: A is a representative image of the vascular status of zebrafish tails in different treatment groups, with the red dashed box indicating the site of thrombus formation; B is a statistical graph of tail arterial flow velocity; compared with group cd41... &&& P <0.001; with CALR del52 Compared to the group, ### P <0.001; with CALR del52 Compared to group +AA *** P <0.001; AA represents arachidonic acid, and ASA represents aspirin.

[0026] Figure 11 It has the structural formula of mycophenolic acid methyl ester.

[0027] Figure 12 Figure showing the effect of methyl mycophenolate on the distribution of erythrocytes in thrombotic zebrafish; In the figure: A is a staining map of zebrafish erythrocytes; B is a statistical chart of the staining area and intensity of erythrocytes in the heart; C is a statistical chart of the staining area and intensity of erythrocytes in the tail; compared with group cd41, & P <0.05, &&& P <0.001; with CALR del52 Compared to the group, ### P <0.001; with CALR del52 Compared to group +AA * P <0.05, ** P <0.01, *** P <0.001.

[0028] Figure 13 The effect of mycophenolate methyl ester on the expression levels of thrombosis-related factors in zebrafish; In the figure: A represents thromboxane A2 (TXA2) content, B represents von Willebrand factor (vWF) content, and C represents D-dimer content; compared with the cd41 group, &&& P <0.001; with CALR del52 Compared to the group, ### P <0.001; with CALR del52 Compared to group +AA ** P <0.01, *** P <0.001. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0030] Unless otherwise specified in the examples, the procedures were performed under standard conditions; reagents or instruments used without a specified manufacturer were all commercially available products.

[0031] 1 Materials and Instruments 1.1 Experimental Reagents RNase-Free Deionized Water (Thermo Fisher Scientific, catalog number A57775), 50×TAEBuffer (Sangon Biotech, catalog number B548101-0500), DL2000 Plus DNA Marker (Novozymes Biotechnology, catalog number MD101-01), Ultra GelRed (Novozymes Biotechnology, catalog number GR501-01), 2×Rapid Taq Master Mix (Novozymes Biotechnology, catalog number P222-01), Agarose (Yisheng Biotechnology Co., Ltd., catalog number 10208ES60), Sodium Chloride (Sangon Biotech, catalog number A610476-0001), FastDigest NotI (Thermo Fisher Scientific, catalog number FD0594), Potassium chloride (Sangon Biotech, catalog number A610440-0500), Calcium chloride dihydrate (Sangon Biotech Co., Ltd., catalog number A610050-0500), Magnesium chloride hexahydrate (Sangon Biotech Co., Ltd., catalog number A601336-0500), Methylene Blue (Sigma-Aldrich Corporation, USA, catalog number M9140), MS-222 (Sigma-Aldrich Corporation, USA, catalog number A5040), 10× DNA Loading Buffer (Novozymes Biotechnology Co., Ltd., catalog number P022-01), mMESSAGE mMACHINE™ SP6 Transcription Kit (Thermo Fisher Scientific, catalog number AM1340), One-Step Cell Genotyping Kit (Nanjing Yaoshunyu Biotechnology Co., Ltd., catalog number K-101-100), YSY Buffer (Nanjing Yaoshunyu Biotechnology Co., Ltd., catalog number K-101-100), Axygen PCR Cleaning Kit (Axygen Biotechnology Co., Ltd., catalog number AP-PCR-50), Minerva SuperFusion Cloning Kit (Everbright, USA) inc, catalog number M2026S), pCS2-transposase plasmid (National Zebrafish Resource Center, catalog number CZP13), Nuclease-free Water (Thermo Fisher Scientific, catalog number AM9937), Lithium Chloride, Arachidonic acid (AA), Aspirin,The following kits were purchased from Jiangsu Enzyme Immunosorbent Assay Co., Ltd.: ASA, 1-Phenyl-2-thiourea (PTU), Mycophenolic Acid Methyl Ester (MAE, CAS No. 31858-66-9), thromboxane A2 (TXA2), von Willebrand factor (vWF), and D-dimer. All three thrombosis-related factor kits were purchased from Jiangsu Enzyme Immunosorbent Assay Co., Ltd.

[0032] 1.2 Experimental Apparatus Zebrafish embryo microinjection instrument IM-300 (Neimo Corporation, Japan), Bori New XP gene amplification instrument TC-XP-G (Hangzhou Bori Technology Co., Ltd.), zebrafish rearing system (Beijing Aisheng Technology Co., Ltd.), horizontal electrophoresis system HE-120 (Shanghai Tianneng Technology Co., Ltd.), Tanon 1600 series multifunctional gel image analysis system (Shanghai Tianneng Life Science Co., Ltd.), Olympus inverted fluorescence microscope (Olympus Ltd., Japan, model SZX16), Ausen Nano-100 micro spectrophotometer (Hangzhou Ausen Instrument Co., Ltd., model Nano-100), low-temperature centrifuge (Eppendorf AG, Germany, model 5428000295), gradient PCR instrument (Thermal Cycler, model BIO-RAD C100), real-time PCR instrument (Roche, Switzerland, model LightCycler). 96) Micro-volume spectrophotometer (ThermoFisher Technology, USA, model NanoDrop™ OneC), ZebraBlood flowmeter v1.3.2 (ViewPoint, France), Zeiss fluorescence microscope (Carl Zeiss GmbH, Germany, model AXIO-V16).

[0033] 1.3 Experimental Animals This experiment used wild-type AB strain zebrafish and transgenic zebrafish with green fluorescently labeled platelets. Tg(cd41: eGFP) Zebrafish can be purchased from the National Zebrafish Resource Center or from commercially available products. Male and female zebrafish should be raised separately under standard conditions of 14 hours of light / 10 hours of darkness at 28 ± 0.5℃, and fed with pelleted food at regular intervals. For egg collection, healthy, sexually mature zebrafish should be placed in a mating tank at a female-to-male ratio of 2:2 or 1:2. Fertilized eggs should be obtained between 10 and 11 am the following day. The embryos should be washed three times with zebrafish culture water, then disinfected with 0.1% methylene blue, and transferred to zebrafish culture water (5 mM NaCl, 0.33 mM CaCl2, 0.33 mM MgSO4•7H2O, and 0.17 mM KCl) and cultured under controlled light at 28 ± 0.5℃.

[0034] 2. Experimental Methods In previous studies, our research team overexpressed [the gene] in zebrafish somatic cells. CALR del52 By combining low-concentration arachidonic acid (RAA) as a thrombotic inducer with short-term induction, a zebrafish model of thrombosis accompanied by thrombosis was successfully constructed. Tg(cd41: eGFP; EF1a: CALR del52 ）， The model was used to screen various compounds for their antithrombotic activity.

[0035] 2.1 Construction of a zebrafish platelet hyperplasia model 2.1.1 Tol2-EF1a-h CALR del52 Design and construction of the IRES2-mcherry-UTRsv40 recombinant expression vector EF1a is a potent and broad-spectrum promoter capable of driving gene expression in various cell types and tissues. Highly conserved in mammals, fish, and other eukaryotes, it is commonly used to construct transgenic animals or cell lines to achieve stable and efficient expression of target genes. IRES2 is an internal ribosome entry site that allows ribosomes to directly bind to mRNA and initiate translation without relying on the 5' cap structure. Its main function is to achieve polycistronic expression, enabling multiple genes to be co-expressed under the regulation of the same promoter. mCherry is a red fluorescent protein with a relatively simple gene sequence, facilitating fusion expression with other genes. Through genetic engineering, the mCherry gene can be linked to the target gene to construct a fusion protein expression vector. After intracellular expression, mCherry acts as a fluorescent tag, visually indicating the location of the protein encoded by the target gene. UTRSV40 is a highly efficient and reliable gene expression regulatory element widely used in animal expression vectors, playing a crucial role in gene expression regulation. It is used to enhance gene expression, improve mRNA stability, and optimize vector design. The Tol2 vector, capable of expressing the mcherry reporter gene throughout zebrafish, was modified using molecular cloning methods, by inserting a gene between the EF1a promoter and the mcherry coding region. CALR The nucleotide sequence after removing 52 bp from exon 9 yields Tol2-EF1a-h. CALR del52 -IRES2-mcherry-UTRsv40 recombinant expression vector. The vector construction was commissioned to Nanjing Yaoshunyu Biotechnology Co., Ltd.

[0036] CALR del52 The nucleotide sequence is shown in SEQ ID NO.1.

[0037] Tol2-EF1a-h CALR del52 The nucleotide sequence of the -IRES2-mcherry-UTRsv40 recombinant expression vector is shown in SEQ ID NO.2.

[0038] 2.1.2 Synthesis of Tol2 transposase mRNA The pCS2-transposase plasmid (purchased from the National Zebrafish Research Center) was linearized with Not1 restriction endonuclease and transcribed using the mMESSAGE mMACHINE™ SP6 (Thermo Fisher Scientific) transcription kit according to the manufacturer's instructions to obtain Tol2 transposase mRNA. Purification was then performed using lithium chloride precipitation, and the Tol2 transposase mRNA was resuspended in 20 μL of nuclease-free water. RNA concentration was determined, and the RNA was stored frozen at −70°C.

[0039] The specific operating steps are as follows: 2.1.2.1 pCS2-transposase plasmid linearization by digestion The pCS2-transposase plasmid was linearized using the Not1 restriction endonuclease. The experimental procedures are shown in Table 1. The reaction conditions were: 37℃ water bath for 2 h. 0.5 µL of the sample was taken for agarose gel electrophoresis (1%) to determine its integrity.

[0040] Table 1 Enzyme digestion reaction system

[0041] 2.1.2.2 Purification of in vitro transcription template The recovered products were collected using a nuclease-free PCR cleaning kit (Axygen). The procedure is as follows: 1. Add 3 volumes of Buffer PCR-A to the linearized plasmid, mix well, transfer to a preparation tube, place the preparation tube in a 2 mL centrifuge tube (provided in the kit), centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0042] 2. Place the preparation tube back into a 2 mL centrifuge tube, add 700 μL Buffer W2, centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0043] 3. Place the preparation tube back into the 2 mL centrifuge tube, add 400 μL Buffer W2, centrifuge at 12,000×g for 1 min, and discard the filtrate.

[0044] 4. Place the preparation tube into a clean 1.5 mL centrifuge tube (provided in the kit), add 30 μL of Eluent to the center of the membrane in the preparation tube, and let it stand at room temperature for 1 min. Centrifuge at 12,000×g for 1 min and collect the eluent.

[0045] 2.1.2.3 In vitro transcription mRNA was synthesized using the mMESSAGE mMACHINE™ SP6 Kit transcription reagent and SP6 transcriptase. The reaction system is shown in Table 2. The reaction conditions were: 37°C for 2 h. After in vitro transcription was completed, 1 µL of TURBO DNase I was added, and the mixture was incubated at 37°C for another 15 min to remove the DNA template.

[0046] Table 2 In vitro transcription reaction system

[0047] 2.1.2.4 Purification of in vitro transcribed mRNA 1. Add 50 μL of lithium chloride precipitate solution to the reaction product tube in 2.1.2.3 and store overnight at −80°C.

[0048] 2. Centrifuge at 12000 g for 10 min at 4℃.

[0049] 3. Carefully discard the supernatant and add 1000 µL of 75% ethanol (prepared with DEPC water). Vortex thoroughly to wash, and gently tap the bottom of the tube to suspend the precipitate.

[0050] 4. Centrifuge at 12000g for 10 min at 4℃, discard the supernatant, being careful not to lose the RNA precipitate.

[0051] 5. Repeat steps 3-4.

[0052] 6. Dry in a fume hood for 1 min, add 20 µL of Nuclease-free water to dissolve the RNA. After complete dissolution, take 1 μL of the sample for gel electrophoresis analysis and spectrophotometer concentration determination. Store the remaining solution at −80℃.

[0053] 2.1.3 Zebrafish microinjection and embryo selection Wild-type AB line zebrafish fertilized eggs were collected using standard methods, and Tol2-EF1a-h CALR del52The -IRES2-mcherry-UTRsv40 recombinant expression vector was mixed with Tol2 transposase mRNA at a final concentration of 200 ng / μL and microinjected into zebrafish fertilized eggs within 15 minutes of fertilization. The injection volume was 1 nL per embryo. The injected zebrafish were treated with phenylthiourea (PTU) to 48 hpf (Hours Post Fertilization) for fluorescence observation. The observation of red fluorescent spots in the zebrafish indicated that the transgenic expression system was functioning normally. Fluorescent zebrafish were selected and raised to adulthood (F0). The F0 cells were then compared with... Tg(cd41:eGFP) Embryos expressing both red and green fluorescence were selected at 48 hpf and cultured to adulthood (F1). F1 males and females were self-crossed to produce F2 generation, and zebrafish expressing red and green fluorescence (i.e., overexpression) were selected. CALR del52 Transgenic zebrafish with green fluorescently labeled platelets Tg (cd41: eGFP; EF1a: CALR del52 ） .

[0054] 2.1.3.1 Extraction of F1 generation transgenic zebrafish genome Five F1 embryos were collected to prepare genomic DNA templates. The genomic DNA templates were prepared using a one-step cell genotyping kit from Nanjing Yaoshunyu Biotechnology Co., Ltd. The reaction conditions were: 65℃ for 30 min, 95℃ for 5 min, 16℃ for 1 min, and 4℃.

[0055] 2.1.3.2 PCR detection of F1 generation transgenic zebrafish genome h was determined by PCR amplification. CALR del52 Whether it integrates into the F1 generation zebrafish genome. PCR amplification system: 20 μL 2×Mastermix (Vazyme), 14 μL ultrapure water, 2 μL forward and reverse (Primer-F and Primer-R) primers (10 μM), and 2 μL genomic DNA template prepared in 2.1.3.1. PCR reaction conditions: 95℃ for 3 min, 34× (95℃ for 15 s, 56℃ for 15 s, 72℃ for 15 s), 72℃ for 5 min, 4℃. Amplification primer sequences are shown in Table 3.

[0056] Table 3 Primer sequences for PCR amplification

[0057] 2.1.3.3 Extraction of F2 generation transgenic zebrafish genome Five F2 generation zebrafish fertilized eggs were collected using standard methods. Genomic DNA templates were prepared using a one-step cell genotyping kit produced by Nanjing Yaoshunyu Biotechnology Co., Ltd. The specific reaction conditions were: 65℃ for 30 min, 95℃ for 5 min, 16℃ for 1 min, and 4℃.

[0058] 2.1.3.4 PCR detection of F2 generation transgenic zebrafish genome Detection of h via PCR amplification CALR del52 Whether it was successfully integrated into the F2 generation zebrafish genome. PCR amplification reaction system (40 μL): 20 μL 2×Mastermix (Vazyme), 14 μL ultrapure water, 2 μL forward and reverse (Primer-F and Primer-R) primers (10 μM), and 2 μL genomic DNA template prepared in 2.1.3.3. The amplified PCR products were sent to GE Sanger for sequencing. The amplification primer sequences are shown in Table 3.

[0059] 2.1.4 Research on Disease Progression in Transgenic Zebrafish use Tg(cd41:eGFP) Zebrafish were used as a blank control to construct a zebrafish model with increased platelets. Tg (cd41:eGFP; EF1a:CALR) del52 ） Phenotypic observation was conducted. Starting from 3 dpf (Day Post Fertilization), 10 juvenile fish were randomly selected and placed under a Zeiss fluorescence microscope and blood flow analyzer. For 5 consecutive days, the number of circulating platelets, the area of ​​platelet aggregation in the tail and fluorescence intensity, changes in tail arterial blood flow velocity, and changes in the content of thrombosis-related factors were detected.

[0060] 2.1.4.1 Experimental Grouping Experimental setup: blank control group Tg(cd41:eGFP) and the transgenic zebrafish model group with primary thrombocytosis Tg (cd41:eGFP; EF1a:CALR) del52 ） Zebrafish with a dpf of 3-7 were selected under a microscope and transferred into a 24-well plate, with 10 zebrafish per well and 3 duplicate wells.

[0061] 2.1.4.2 Circulating platelet count detection Select Tg (cd41: eGFP; EF1a: CALR del52 ） and Tg(cd41:eGFP)Zebrafish strains were used, and the experimental groups are described in section 2.1.4.1. Ten juvenile zebrafish were randomly selected from each group and washed three times with zebrafish culture water. The number of circulating platelets flowing through the tail of the zebrafish within 15 seconds was recorded under an Olympus inverted fluorescence microscope, and statistical analysis was performed.

[0062] 2.1.4.3 Detection of platelet aggregation area and fluorescence intensity at the tail end use Tg (cd41: eGFP; EF1a: CALR del52 ） and Tg(cd41:eGFP) Zebrafish strains were used, and the experimental groups are described in section 2.1.4.1. Ten juvenile zebrafish were randomly selected from each group, and images of the zebrafish were acquired under an Olympus inverted fluorescence microscope. The platelet aggregation area and fluorescence intensity in the tail of the zebrafish were measured and calculated using Image-Pro Plus software, and statistical analysis was performed.

[0063] 2.1.4.4 Detection of blood flow velocity in the tail artery Select Tg (cd41: eGFP; EF1a: CALR del52 ） and Tg(cd41:eGFP) Zebrafish strains were used, and the experimental groups are described in section 2.1.4.1. Ten juvenile zebrafish were randomly selected from each group, and the blood flow in the caudal artery of the zebrafish was recorded for 15 seconds using a blood flow meter. The blood flow velocity in the caudal artery of each group was statistically analyzed using ZebraBlood software.

[0064] 2.1.4.5 Detection of thrombosis-related factor levels collect Tg(cd41:eGFP) Blank control group and Tg (cd41: eGFP; EF1a: CALR del52 ） Zebrafish in the thrombocytosis model group were washed three times with phosphate-buffered saline (PBSS). Following the manufacturer's instructions, PBSS (0.01 M, pH 7.4) was added, and the tissue homogenate was obtained by sonication. According to the manufacturer's instructions, thromboxane A2 (TXA2), von Willebrand factor (vWF), tissue factor (TF), and D-dimer, all closely related to thrombosis, were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Sixty zebrafish were used in each group, and each experiment was performed three times.

[0065] 2.2 Construction of a zebrafish model of thrombocytosis accompanied by thrombosis 2.2.1 Determination of Arachidonic Acid Treatment Concentration and Treatment Time Select 3-4 dpf Tg (cd41: eGFP; EF1a: CALRdel52 ） and Tg(cd41:eGFP) Transgenic zebrafish, with a blank control group (cd41 / CALR) set up in the experiment. del52 -Control), Arachidonic acid (AA) treatment group (cd41 / CALR) del52 (+different concentrations of AA), 10 zebrafish per well, with 3 replicates. The blank control group was treated with zebrafish culture water, while the arachidonic acid treatment groups were treated with different concentrations of arachidonic acid solution. All groups were incubated at 28 ± 0.5℃ in the dark for different durations. After arachidonic acid treatment, 15 juvenile zebrafish were randomly selected from each group, washed three times with zebrafish culture water, and the blood flow in the caudal artery was recorded for 15 seconds using a blood flow meter. The blood flow velocity in the caudal artery of each group was statistically analyzed using ZebraBlood software.

[0066] 2.3 Applicability Validation of Antithrombotic Drug Screening in a Zebrafish Model with Thrombosis and Thrombosis 2.3.1 Experimental Grouping Zebrafish with a 3-4 dpf growth rate were selected under a microscope and transferred into 24-well plates. Two blank control groups (cd41, CALR) were set up for the experiment. del52 ), thrombosis model group (CALR) del52 +AA), positive drug aspirin (ASA) group (CALR) del52 (+AA+ASA), 10 strips per well, with 3 replicates. The blank control group and thrombosis model group were treated with zebrafish culture water, while the positive control group was treated with 125 μM aspirin solution. Zebrafish in all groups were incubated at 28 ± 0.5℃ for 6 h. The solution was then aspirated. The blank control group was treated with zebrafish culture water, while the other groups were treated with arachidonic acid (AA) solution at a final concentration of 20 μM and kept in the dark for 20 min.

[0067] 2.3.2 Circulating platelet count detection Select Tg (cd41: eGFP; EF1a: CALR del52 ） and Tg(cd41:eGFP) For transgenic zebrafish, the experimental grouping and drug treatment are described in 2.3.1. The experimental procedure is the same as in 2.1.4.2.

[0068] 2.3.3 Detection of platelet aggregation area and fluorescence intensity at the tail end use Tg (cd41: eGFP; EF1a: CALR del52 ） and Tg(cd41:eGFP) For transgenic zebrafish, the experimental grouping and drug treatment are described in 2.3.1. The experimental procedure is the same as in 2.1.4.3.

[0069] 2.3.4 Detection of blood flow velocity in the tail artery Select Tg (cd41: eGFP; EF1a: CALR del52 ） and Tg(cd41:eGFP) For zebrafish strains, experimental grouping and drug treatment are described in 2.3.1. Experimental procedures are the same as in 2.1.4.4.

[0070] 2.4 Evaluation of the antithrombotic activity of mycophenolate mofetil in a zebrafish model with thrombocytosis and thrombosis 2.4.1 Drug grouping Pick 3 dpf Tg (cd41: eGFP; EF1a: CALR del52 ） and Tg(cd41:eGFP) Zebrafish were transferred into 24-well plates. Two blank control groups were set up in the experiment (cd41, CALR). del52 ), thrombosis model group (CALR) del52 +AA), positive control group (CALR) del52 +AA+ASA), methyl mycophenolate treatment group (CALR) del52 Zebrafish were cultured in a solution containing AA and MAE (+AA+MAE), with 10 strips per well and 3 replicates. The blank control group and thrombosis model group were treated with water for zebrafish culture, the positive control group with 125 μM aspirin solution, and the mycophenolate mofetil treatment groups with 5, 10, and 20 μM MAE solution, respectively. All zebrafish were incubated at 28 ± 0.5℃ for 6 h. After 6 h, except for the blank control group, all other groups were treated with AA solution to a final concentration of 20 μM and kept in the dark for 20 min.

[0071] 2.4.2 Detection of erythrocyte staining area and staining intensity After treatment, zebrafish in each group were stained with o-anisidine. Ten zebrafish were randomly selected from each group, and images of the zebrafish were acquired under a Zeiss fluorescence microscope. Image-Pro Plus software was used to measure and calculate the staining area and staining intensity of red blood cells in the heart and tail of the zebrafish.

[0072] 2.4.3 Detection of thrombosis-related factor levels After treatment, zebrafish from each group were collected, and the levels of thromboxane A2 (TXA2), von Willebrand factor (vWF), and D-dimer, which are closely related to thrombosis, were detected using an enzyme-linked immunosorbent assay (ELISA) kit according to the instructions.

[0073] 2.4.4 Circulating Platelet Count Detection The experimental procedure is the same as in 2.1.4.2.

[0074] 2.4.5 Detection of platelet aggregation area and fluorescence intensity at the tail end The experimental procedure is the same as in 2.1.4.3.

[0075] 3. Experimental Results 3.1 Construction of a zebrafish platelet hyperplasia model 3.1.1 Tol2-EF1a-h CALR del52 Construction results of the -IRES2-mcherry-UTRsv40 recombinant expression vector Tol2-EF1a-h CALR del52 The map of the -IRES2-mcherry-UTRsv40 recombinant expression vector is shown below. Figure 1 As shown, its sequence is SEQ ID NO.2.

[0076] 3.1.2 Tg (cd41: eGFP; EF1a: CALR del52 ） Zebrafish screening and identification results 3.1.2.1 Tg (cd41: eGFP; EF1a: CALR del52 ） F1 generation screening and identification Choose those with red fluorescence CALR Genetically mutated zebrafish were raised to adulthood (F0), and then F0 was compared with... Tg(cd41:eGFP) Hybridization was performed, and zebrafish embryos expressing both red and green fluorescence were selected at 48 hpf (F1). Figure 2 As shown in Figure A, the development of the organism to 48 hours was observed under an inverted fluorescence microscope. CALR In F1 generation zebrafish with gene mutation, red and green fluorescent spots were found in the heart area, indicating that the transgenic expression system can function normally and that platelets are specifically labeled with green fluorescent protein. Figure 2 B is CALR Electrophoresis image of PCR amplification products from F1 generation of mutant zebrafish, showing the PCR amplification covering part of the EF1a promoter sequence and part of the EF1a promoter sequence. CALR52 Sequence. The results showed that the mutant gene was successfully transferred into the zebrafish genome. Figure 2 The C-value indicates that Tol2-EF1a-h can be detected by sequencing the F1 generation. CALR del52 The key elements of the -IRES2-mcherry-UTRsv40 recombinant expression vector demonstrate that the mutated gene is heritable.

[0077] 3.1.2.2 Tg (cd41: eGFP; EF1a: CALR del52 ） F2 generation screening and identification Transgenic F1 zebrafish exhibiting red and green fluorescence were raised to adulthood and then self-crossed to obtain the F2 generation. The genome sequence of the F2 generation zebrafish was then determined. Figure 3 As shown, Tol2-EF1a-h can be detected by sequencing the F2 generation. CALR del52 The key elements of the -IRES2-mcherry-UTRsv40 recombinant expression vector indicate stable inheritance. Tg(cd41:eGFP;EF1a:) CALR del52 ） The transgenic zebrafish has been successfully constructed.

[0078] 3.1.3 Transgenic Zebrafish Tg (cd41: eGFP; EF1a: CALR del52 ） Disease progression research results 3.1.3.1 Changes in circulating platelet count Platelet count and platelet aggregation function are important indicators for assessing platelet function and activation status, and help to determine the risk of thrombosis. Figure 4 visible, Tg (cd41: eGFP; EF1a: CALR del52 ） Transgenic zebrafish and control group Tg (cd41:eGFP) Compared to zebrafish, the number of circulating platelets increased at 3 dpf, 4 dpf, 5 dpf and 6 dpf, with a significant difference at 5 dpf.

[0079] 3.1.3.2 Changes in platelet aggregation area and fluorescence intensity at the tail end Figure 5 visible, Tg (cd41: eGFP; EF1a: CALR del52 ） Transgenic zebrafish and control group Tg(cd41:eGFP) Compared to zebrafish, the area of ​​platelet aggregation in the tail increased and the fluorescence intensity increased at 3 dpf and 4 dpf. As the number of days increased, the platelets aggregated in the tail gradually entered the systemic circulation after 4 dpf, and there were fewer platelets aggregated in the tail of zebrafish at 5 dpf and beyond.

[0080] 3.1.3.3 Changes in blood flow velocity in the tail artery Figure 6 It can be seen that 3 dpf and 4 dpf Tg (cd41: eGFP; EF1a: CALR del52 ） Transgenic zebrafish and control group Tg (cd41:eGFP) Compared to zebrafish, the blood flow velocity in the caudal artery was significantly slower, but no spontaneous thrombus formation occurred; the caudal artery blood flow velocity recovered at 5-7 days post-flood (dpf), suggesting... CALR del52The blood circulation disorder in overexpressing transgenic zebrafish was self-healed to a certain extent, meaning that the blood flow velocity returned to normal.

[0081] 3.1.3.4 Changes in the levels of thrombosis-related factors Increased platelet count leads to increased blood viscosity, microcirculatory disturbances, and the release of procoagulant substances such as thromboxane A2 (TXA2) from platelets. These substances can activate other platelets, promoting platelet aggregation and increasing the probability of thrombus formation. Vonoblastic hemophilia factor (vWF) is an important plasma component that plays a hemostatic role by binding to platelets and collagen fibers to form thrombi. Tissue factor (TF) is the receptor for coagulation factor VII, responsible for initiating the extrinsic coagulation cascade and promoting fibrin clot formation. D-dimer is produced after the degradation of cross-linked fibrin, indicating intravascular coagulation and serving as a marker of overall activation of the coagulation and fibrinolytic systems. Therefore, this study examined transgenic zebrafish at 3-7 dpf and 14 dpf. Tg (cd41: eGFP; EF1a: CALR del52 ） Changes in the levels of TXA2, vWF, TF, and D-dimer in the body. Results showed Tg (cd41: eGFP; EF1a: CALR del52 ） The levels of thrombosis-related factors in transgenic zebrafish were significantly higher than those in other species. Tg(cd41:eGFP) Blank control group ( Figure 7 ).

[0082] The above disease progression research results show that, compared with the control group Tg(cd41:eGFP) Compared to zebrafish, Tg(cd41: eGFP; EF1a: CALR del52 ） Transgenic zebrafish showed an increase in circulating platelet count at 3-6 days post-flop (dpf), with a significant difference at 5 dpf. At 3 and 4 dpf, the area and fluorescence intensity of platelet aggregation in the tail increased; after 4 dpf, these tail-aggregated platelets gradually entered systemic circulation, with fewer at 5 dpf and beyond. At 3 and 4 dpf, the blood flow velocity in the tail artery significantly decreased without spontaneous thrombosis; the blood flow velocity recovered at 5-7 dpf, suggesting... Tg(cd41: eGFP; EF1a: CALR del52 ） Zebrafish exhibited spontaneous remission. Furthermore, this study examined the expression levels of thrombosis-related factors in zebrafish. It was found that... Tg(cd41: eGFP; EF1a: CALR del52 ） The levels of thrombosis-related factors in zebrafish were significantly higher than those in other fish. Tg(cd41: eGFP) In summary, this study selected patients with a 3-4 day post-thrombotic risk (dpf) of higher risk. Tg(cd41: eGFP; EF1a: CALR del52 ）Construction of a zebrafish model of thrombocytosis accompanied by thrombosis.

[0083] 3.2 Construction of a zebrafish model of thrombocytosis accompanied by thrombosis 3.2.1 Effects of different concentrations of arachidonic acid treatment for different durations on blood flow velocity in zebrafish In this study, the zebrafish thrombocytosis model showed a significant decrease in blood flow velocity, but no spontaneous thrombosis was observed. Thrombosis is a complex pathological process, triggered not only by intrinsic factors such as coagulation system abnormalities, platelet activation, and endothelial injury, but also by exogenous factors such as chemical substances, hemodynamic changes, and inflammatory stimulation. Therefore, this study used exogenous arachidonic acid (AA) to treat transgenic zebrafish with thrombocytosis to construct a thrombosis model. Different concentrations of arachidonic acid were used to treat 3-4 dpf transgenic zebrafish. Tg(cd41: eGFP; EF1a: CALR del52 ） Different times. Figure 8 visible, Tg(cd41: eGFP) The blood flow velocity in the tail artery of zebrafish after treatment with 20 μM arachidonic acid for 20 min was compared with... Tg(cd41: eGFP) There was no significant difference compared to the control group. Under the same treatment concentration and treatment time conditions, CALR del52 Overexpression of zebrafish Tg(cd41: eGFP; EF1a: CALR del52 ） The blood flow velocity in the tail artery was significantly lower than Tg(cd41: eGFP) In the control group, local blood flow obstruction and thrombus formation were observed. In the treatment groups with higher concentrations (40 μM, 60 μM, 80 μM) and longer treatment times (40 min, 60 min), Tg(cd41: eGFP; EF1a: CALR del52 ） The blood flow velocity in the tail artery was significantly lower than that in other arteries. Tg (cd41: eGFP) Control group. Therefore, treatment with 20-80 μM arachidonic acid for 20-60 min was used as the control group for transgenic zebrafish in this study. Tg(cd41: eGFP; EF1a: CALR del52 ） A method for thrombosis modeling was used to construct a zebrafish model of thrombosis accompanied by thrombocytosis. Previous literature often used arachidonic acid at concentrations of 80 μM or higher to treat non-thrombotic platelets. CALR del52 A thrombosis model was induced in zebrafish by overexpression for more than 1 hour. In this study... CALR del52 Overexpression of arachidonic acid, which is required for thrombosis in zebrafish, requires lower concentrations and shorter treatment times, enabling the screening of antithrombotic compounds at lower cost and higher efficiency.

[0084] 3.3 Applicability Validation of Aspirin (ASA), an Antithrombotic Therapy Drug, in a Zebrafish Model of Thrombosis-Related Thrombotic Disease with Thrombosis To verify the applicability of the established zebrafish model of thrombocytosis accompanied by thrombosis for drug screening, aspirin, an internationally recognized antithrombotic drug, was selected to test its antithrombotic effect on the model. The core mechanism of aspirin's antithrombotic effect is the inhibition of platelet aggregation. This is achieved by irreversibly inhibiting cyclooxygenase (COX)-1 and (COX)-2, preventing the production of TXA2 from arachidonic acid, thereby blocking TXA2-induced platelet aggregation and exerting an antithrombotic effect. In this study, aspirin was used to treat juvenile zebrafish in the thrombocytosis-associated disease model. The antithrombotic activity of aspirin was assessed by detecting indicators such as circulating platelet count, tail platelet aggregation area and fluorescence intensity, and tail artery blood flow velocity to verify whether this model is suitable for antithrombotic drug screening.

[0085] 3.3.1 Effects of aspirin on circulating platelet count, tail platelet aggregation area, and fluorescence intensity Arachidonic acid can cause thrombosis by inducing platelet aggregation. Figure 9 The results showed that, compared with the blank control group, platelet aggregation occurred in the tail of zebrafish in the thrombosis model group, the number of circulating platelets was significantly reduced, and the area of ​​aggregated platelets and fluorescence intensity in the tail were significantly increased. Compared with the thrombosis model group, the number of circulating platelets in zebrafish in the positive drug aspirin group was significantly increased, and the area of ​​aggregated platelets and fluorescence intensity in the tail were significantly reduced.

[0086] 3.3.2 Effect of Aspirin on Blood Flow Velocity in the Caudal Artery An increased platelet count increases blood viscosity, leading to slower blood flow. Slower blood flow makes it easier for formed elements in the blood to deposit on the blood vessel walls, causing platelets to adhere to vascular endothelial cells and aggregate, thereby promoting thrombus formation. Figure 10 It can be seen that, compared with the blank control group, CALR del52 +AA group: The blood flow velocity in the caudal artery of zebrafish was significantly slowed, and thrombi formed locally in the arteries and veins, indicating that the thrombosis model was successfully established. CALR del52 The caudal artery flow velocity in the +AA+ASA group of zebrafish was lower than that in the CALR group. del52 The significant increase in AA levels indicates that aspirin has antithrombotic efficacy in a zebrafish model of thrombocytosis accompanied by thrombosis.

[0087] 3.4 Antithrombotic effect of mycophenolate methyl ester like Figure 12As shown, compared with the blank control group, the thrombosis model group showed a significant decrease in the staining area and intensity of cardiac erythrocytes, and a significant increase in the staining area and intensity of tail erythrocytes, with erythrocyte aggregation in the main vein. After treatment with the positive control drug aspirin, the staining area and intensity of zebrafish cardiac erythrocytes were significantly increased compared with the thrombosis model group, while the staining area and intensity of tail erythrocytes decreased, and erythrocyte aggregation in the main vein was alleviated. Compared with the thrombosis model group, the zebrafish treated with methyl mycophenolate mofetil showed a significant increase in the staining area and intensity of cardiac erythrocytes, and a significant decrease in the staining area and intensity of tail erythrocytes, while effectively alleviating erythrocyte aggregation caused by aspirin in the main vein; the antithrombotic effect of 10 μM methyl mycophenolate mofetil was superior to that of 125 μM positive control drug aspirin.

[0088] like Figure 13 The results showed that, compared with the blank control group, the zebrafish in the model group had significantly increased levels of thrombosis-related factors TXA2, vWF, and D-dimer, and mycophenolate mofetil could significantly reduce the expression of AA-induced thrombosis-related factors and inhibit thrombus formation.

[0089] Compared with the blank control group, the thrombosis model group of zebrafish showed increased platelet aggregation in the tail, a significantly increased tail platelet aggregation area, and enhanced fluorescence intensity, while the number of circulating platelets was significantly reduced. Methyl mycophenolate mofetil treatment could alleviate platelet aggregation in thrombotic zebrafish, reduce the tail platelet aggregation area and fluorescence intensity, and increase the number of circulating platelets, demonstrating an antithrombotic effect.

[0090] The above results indicate that methyl mycophenolate mofetil has significant antithrombotic activity.

[0091] Our research team overexpressed [the gene] in zebrafish somatic cells. CALR del52 By combining low-concentration arachidonic acid (RAA) as a thrombotic inducer with short-term induction, a zebrafish model of thrombosis accompanied by thrombosis was successfully constructed. Tg(cd41: eGFP; EF1a: CALR del52 ） Aspirin is a commonly used antithrombotic drug in clinical practice. In a zebrafish model of thrombocytosis accompanied by thrombosis, aspirin treatment significantly increased the number of circulating platelets, significantly reduced the platelet aggregation area and fluorescence intensity in the tail, significantly restored arachidonic acid-induced circulatory disturbances, and significantly increased the blood flow velocity in the tail artery. The antithrombotic therapeutic effect of aspirin was significant and highly consistent with its efficacy in human clinical treatment, verifying that the zebrafish model of thrombocytosis accompanied by thrombosis provided by this invention can be used for screening antithrombotic drugs.

[0092] Based on the zebrafish model of thrombosis accompanied by thrombosis provided in this invention, a systematic evaluation of the antithrombotic activity of mycophenolate mofetil was conducted. Experimental results showed that mycophenolate mofetil has a significant antithrombotic effect, reducing platelet aggregation in thrombotic zebrafish, decreasing the platelet aggregation area and fluorescence intensity in the tail region, and increasing the circulating platelet count. It can significantly reduce abnormal erythrocyte aggregation in the tail region of thrombotic zebrafish, effectively alleviating arachidonic acid-induced erythrocyte aggregation in the main vein, and inhibiting the expression of thrombosis-related factors such as TXA2, vWF, and D-dimer. Simultaneously, this compound can increase venous return, thereby improving a series of pathological processes induced by thrombosis, suggesting its potential as a candidate drug for the treatment of thrombotic diseases. The research results of this invention provide important experimental evidence and theoretical support for the development of safe and effective novel thrombotic therapeutic drugs.

Claims

1. Application of methyl mycophenolate in the preparation of antithrombotic drugs.

2. The application as described in claim 1, characterized in that, The drug contains one or more pharmaceutically acceptable carriers or excipients.

3. The application as described in claim 2, characterized in that, The excipient is at least one of the following: a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, or a lubricant.

4. The application as described in claim 1, characterized in that, The dosage form of the drug is capsule, pill, tablet, oral liquid, granule, tincture or injection.

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