Application of CDC20 inhibitor in preparation of medicine for preventing or treating aortic dissection

Abstract

The invention discloses application of a CDC20 inhibitor in preparation of a medicine for preventing or treating aortic dissection, and belongs to the field of biological medicine. A mouse aortic dissection model is constructed by combining 3-aortic acid aminopropionitrile ester with angiotensin II, a CDC20 inhibitor Apcin is given for treatment, and experiments find that by inhibiting CDC20 activity through Apcin, the occurrence rate and the lesion area of the mouse aortic dissection can be remarkably reduced, the vascular degenerative change in the lesion of the mouse aortic dissection can be remarkably improved, and the mouse aortic dissection model can be used for treating the aortic dissection of the mouse. Inflammatory macrophage infiltration in the aortic dissection lesion of the mouse is also obviously reduced; the results show that the mouse aortic dissection process can be delayed by inhibiting CDC20. The CDC20 inhibitor (Apcin or similar drugs) is further researched and optimized, so that a novel treatment drug for the aortic dissection is expected to be developed, and the CDC20 inhibitor has important significance in improving early intervention and prognosis of clinical aortic dissection patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a CDC20 inhibitor in preparing a medicament for preventing or treating aortic dissection. Background Art

[0002] The cell cycle refers to the entire process that a cell goes through from the completion of one division to the end of the next division, including four phases: G1 phase (pre-DNA synthesis phase), S phase (DNA synthesis phase), G2 phase (late DNA synthesis phase) and M phase (mitosis phase). This process is a basic component of cell life activities and plays a vital role in cell growth, proliferation, and tissue renewal and repair. Cell cycle regulation guides cells through various phases such as G1 phase, S phase, G2 phase and M phase in an orderly manner, thereby achieving accurate replication of genetic material and orderly cell division. In each stage, key molecules such as specific cyclins, cyclin dependent kinases (CDKs) and their inhibitory proteins (CKIs) play a precise regulatory role to ensure the correct occurrence and timely conversion of cell cycle events. In addition to the above-mentioned Cyclins, CDKs and CKIs, other molecules such as specific transcription factors and signal pathway molecules are also involved in the regulation of the cell cycle. Together with Cyclins, CDKs and CKIs, they form a complex and sophisticated regulatory network that jointly maintains the normal operation of the cell cycle, which is crucial for maintaining the homeostasis of organisms.

[0003] In the cardiovascular system, abnormal cell cycle regulation may cause an imbalance in the proliferation and apoptosis of key cells such as endothelial cells, smooth muscle cells, cardiomyocytes, and fibroblasts, and may also cause abnormal activation of immune inflammatory cells during damage repair, leading to a series of pathological changes such as atherosclerosis, vascular remodeling, and myocardial fibrosis. Therefore, in-depth exploration of the pathophysiological process and regulatory mechanism of the cell cycle will not only help to clarify the pathogenesis of cardiovascular diseases, but also open up new ways for their prevention and treatment.

[0004] Although increasing research evidence shows that cell cycle regulation plays a key role in maintaining cardiovascular homeostasis and injury repair, whether it is involved in the occurrence and development of aortic dissection and its potential regulatory mechanism remain unclear and need to be further elucidated. Cell division cycle 20 (CDC20), as an important activator of the anaphase-promoting complex / cyclosome (APC / C), can bind to APC / C to form the APC / Cdc20 complex, thereby activating the E3 ligase activity of APC / C and mediating the ubiquitination and degradation of specific proteins in the cell cycle. At the beginning of mitosis, CDC20 binds to APC / C and mediates the ubiquitination-dependent degradation of Cyclin B protein by APC / C, resulting in a decrease in the activity of CDK1 (Cyclin Dependent Kinase 1), which in turn triggers the cell to enter the M phase of mitosis from the G2 phase. In the late stage of mitosis, CDC20 continues to bind to APC / C and mediates the degradation of proteins such as Securin and CyclinA. The degradation of Securin is a key step to ensure the accurate separation of sister chromatids, because it removes the inhibition of separase, allowing chromosomes to separate; while the degradation of CyclinA contributes to the smooth completion of mitosis and the exit of the cell cycle. The function of CDC20 is not limited to the regulation of the cell cycle, it is also involved in a variety of biological processes such as apoptosis, DNA damage response, maintenance of stem cell characteristics, and immune cell infiltration. In the field of oncology, CDC20 has attracted widespread attention due to its high expression in a variety of tumors and its association with tumor development. In addition, abnormal expression of CDC20 is associated with poor prognosis in a variety of malignant tumors, making it a potential target for cancer treatment.

[0005] However, whether CDC20 can serve as a potential drug target for the prevention and treatment of aortic dissection remains unclear. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a CDC20 inhibitor in the preparation of a drug for preventing or treating aortic dissection, so as to solve the problems existing in the above-mentioned prior art. The CDC20 inhibitor Apcin provided by the present invention can effectively inhibit the activity of CDC20 and delay the progression of aortic dissection in mice.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides use of a CDC20 inhibitor in preparing a medicament for preventing or treating aortic dissection.

[0009] Preferably, the CDC20 inhibitor is Apcin.

[0010] Preferably, the aortic dissection is aortic dissection induced by 3-fumaric acid aminopropionitrile combined with angiotensin II.

[0011] Preferably, the drug achieves the effect of preventing or treating aortic dissection by reducing the incidence and lesion area of ​​aortic dissection, improving vascular degenerative changes and inflammatory macrophage infiltration in aortic dissection lesions.

[0012] The present invention also provides a medicine for preventing or treating aortic dissection, which comprises a CDC20 inhibitor and pharmaceutically acceptable auxiliary materials.

[0013] Preferably, the CDC20 inhibitor is Apcin.

[0014] Preferably, the dosage form of the drug includes injection, powder, tablet, granule, capsule, solution, emulsion and suspension.

[0015] The present invention discloses the following technical effects:

[0016] The present invention analyzes the human aortic dissection GEO database and finds that CDC20 is significantly increased in human aortic dissection lesions; on this basis, the present invention describes the potential role of CDC20 in the mouse aortic dissection model for the first time. First, the present invention searches the mouse aortic dissection GEO database and confirms that CDC20 is significantly increased in the mouse aortic dissection model constructed by 3-fumaric acid aminopropionitrile (BAPN) combined with angiotensin II; in order to exclude the possible influence of angiotensin II on the expression of CDC20, the present invention also confirms that CDC20 is also significantly increased through a mouse aortic dissection model induced by BAPN alone. The above results show that abnormal increase of CDC20 is a common pathological feature in aortic dissection lesions. In addition, whether it is the mouse aortic dissection model induced by BAPN alone or in combination with angiotensin II, it simulates the pathological phenomenon of abnormal increase of CDC20 in human aortic dissection lesions well, providing a reliable model for subsequent pharmacodynamic verification.

[0017] Next, the present invention administered Apcin, a specific inhibitor of CDC20, to a mouse aortic dissection model constructed by BAPN combined with angiotensin II, and confirmed that Apcin inhibited CDC20 activity and could significantly reduce the incidence and lesion area of ​​aortic dissection in mice. Not only that, Apcin inhibited CDC20 activity and also significantly improved vascular degenerative changes in aortic dissection lesions in mice, including elastic fiber degradation, smooth muscle loss, and extracellular matrix degradation. In addition, Apcin inhibited CDC20 activity and also significantly reduced inflammatory macrophage infiltration in aortic dissection lesions in mice. Our results suggest that CDC20 plays an important pathogenic role in aortic dissection lesions, and inhibiting CDC20 can delay the progression of aortic dissection in mice. CDC20 may serve as a potential drug target for the clinical prevention and treatment of aortic dissection.

[0018] The present invention is not only a further verification of the abnormal increase of CDC20 in aortic dissection lesions described in previously published academic papers, but more importantly, it is the first time to confirm in an animal model that the increase of CDC20 is not only a pathological change of aortic dissection but also plays an important pathogenic role in the disease process. CDC20 may be a potential drug target for the clinical prevention and treatment of aortic dissection. In the future, through further research and optimization of CDC20 inhibitors (Apcin or similar drugs), it is expected to develop new therapeutic drugs for aortic dissection, which is of great significance to improve the early intervention and prognosis of clinical patients with aortic dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1This is a graph showing the results of aortic transcriptome data analysis in the human aortic dissection GEO dataset GSE52093; A is the classification result of differentially expressed genes, Focal adhesion is focal adhesion, Regulation of actin cytoskeleton is actin cytoskeleton regulation, Proteoglycans in cancer is proteoglycans in tumors, Axon guidance is axon guidance, Cell cycle is cell cycle, Neurotrophin signaling pathway is neurotrophin signaling pathway, ECM-receptor interaction is extracellular matrix-receptor interaction, Small cell lung cancer is small cell lung cancer, Central carbon metabolism in cancer is central carbon metabolism in cancer, DNA replication is DNA replication, Generation is gene proportion, P adj is the corrected P value, and counts is count; B is the display result of the differentially expressed gene volcano map, Up is up-regulated, Not sig is not significantly changed, Down is down-regulated, and P.adj is the corrected P value;

[0021] Figure 2 Figure 2 is a differential expression diagram of CDC20 in mouse aortic dissection models; A is the statistical result of CDC20 gene expression in the aorta in the mouse aortic dissection GEO dataset GSE116434, CDC20 gene expression is CDC20 gene expression, Normal is normal mouse, AAD is aortic dissection mouse, ** indicates P<0.01; B is the vascular appearance of the mouse aortic dissection model induced by BAPN and the results of RT-qPCR detection, CTL is the control group, BAPN is the aortic dissection model group constructed by 3-fumaric acid aminopropionitrile, CDC20 gene expression is CDC20 gene expression, * indicates P<0.05; C is the immunohistochemistry result of the cross-section of the thoracic aorta, CTL is the control group, BAPN is the aortic dissection model group constructed by 3-fumaric acid aminopropionitrile, CDC20 positive nuclei are CDC20 positive cell nuclei, * indicates P<0.05;

[0022] Figure 3Figure 2 is the result of Apcin inhibiting aortic dissection in mice; Apcin is the drug-treated group, and Vehicle is the solvent control group; A is the drug-treated regimen, days on BAPN (1 g / kg / d) treatment is the number of days of feeding with 3-fumaric acid aminopropionitrile (at a dose of 1 g / kg / d), AngⅡ is angiotensin II, and Harvest is the harvested material; B is the statistical result of mouse survival, Percent survival is the percentage of survival rate, and ns indicates no significant difference; C is a representative appearance of mouse aorta; D is the statistical result of aortic dissection, AAD incidence is the incidence of aortic dissection, Normal is a normal mouse, AAD is a mouse with aortic dissection, and *** indicates P < 0.001; E is the statistical result of aortic rupture, Percent rupture is the rupture rate, No rupture is no rupture, and Rupture is ruptured; F is the statistical result of aortic lesion area, Dissected segment is the segment with dissected lesions, full length is the total length of the aorta, * indicates P < 0.05; G is the statistical result of the maximum diameter of each segment of the aorta, Max diameter is the maximum diameter, Ascending is the ascending aorta segment, Thoracic is the thoracic aorta segment, Abdominal is the abdominal aorta segment, * indicates P < 0.05;

[0023] Figure 4 Figure 1 is the experimental result of Apcin inhibiting the degenerative changes of mouse aorta; wherein, Apcin is the drug-treated group, and Vehicle is the solvent control group; A is the Verhoeff elastic fiber (EVG) staining result, Elastin score is the elastic fiber score, * indicates P<0.05; B is the α-smooth muscle actin (α-SMA) immunohistochemical staining result, α-SMA+ / Total area is the percentage of α-SMA positive area in the total area of ​​vascular ring, ** indicates P<0.01; C is the matrix metalloproteinase 2 (MMP2) immunohistochemical staining result, MMP2+ / Total area is the percentage of MMP2 positive area in the total area of ​​vascular ring, *** indicates P<0.001; D is the matrix metalloproteinase 9 (MMP9) immunohistochemical staining result, MMP9+ / Total area is the percentage of MMP9 positive area in the total area of ​​vascular ring, *** indicates P<0.001;

[0024] Figure 5The figure shows the results of Apcin inhibiting inflammatory cell infiltration in mouse aortic dissection lesions; wherein, Apcin is the drug-treated group, and Vehicle is the solvent control group; A is the result of CD68 immunohistochemistry staining, EVG is the Verhoeff elastic fiber staining method; B is the statistical result of CD68 positive area, CD68+ / Total area is the percentage of CD68 positive area to the total area of ​​vascular ring, ** indicates P<0.01. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The CDC20 inhibitor selected in the present invention is Apcin, with CAS registration number 300815-04-7, and the structural formula is as follows:

[0031]

[0032] Apcin, as a ligand for CDC20, is a competitive APC / C Cdc20 Apcin is an inhibitor of E3 ligase activity in the complex. Apcin binds to CDC20 by occupying the D-box binding pocket on the side of the WD40 domain, preventing substrate recognition and competitively inhibiting APC / C-mediated ubiquitination and degradation of protein substrates.

[0033] Example 1

[0034] 1. Main Materials and Reagents

[0035] The embodiments described in the present invention are all conventional biomedical experimental methods. The main materials and reagents involved are shown in Table 1 and are all commercially available.

[0036] Table 1 Main materials and reagents used in the present invention

[0037]

[0038]

[0039] 2. Experimental Methods

[0040] 2.1 Analysis of the GEO database of human aortic dissection

[0041] The aortic dissection GEO database was retrieved through the National Center for Biotechnology Information (NCBI) of the United States, and the whole genome expression profiles of diseased aortic samples and normal control aortic samples of patients with aortic dissection were extracted from the GSE52093 dataset "Genome-wide analysis of gene expression in the ascending aorta in patients with acute Stanford type A aortic dissection". The differentially expressed genes DEGs were screened according to the standards of P value less than 0.05 and FC value greater than 1.5, and KEGG analysis and volcano plot were performed.

[0042] 2.2 Experimental animals

[0043] C57BL / 6J wild-type mice were obtained from Liaoning Changsheng Biotechnology Co., Ltd. and were raised in the SPF animal room of Dalian Medical University. All mice were housed in IVC cages and given free access to water and food. All experimental operations in this study were supervised and approved by the Experimental Animal Management Committee of Dalian Medical University and complied with the "Guide to Laboratory Animal Care and Practical Practice" formulated by the National Institutes of Health (NIH).

[0044] 2.3 Induction of aortic dissection in mice

[0045] Male C57BL / 6J mice aged 3-4 weeks were selected and given 3-fumaric acid aminopropionitrile (BAPN) (1 g / kg body weight per day) in drinking water. Angiotensin II (50 μg / kg body weight) was injected subcutaneously for 3 consecutive days starting from the 21st day, and the death of mice was recorded. BAPN treatment was continued until the end of the 28th day, when the samples were collected.

[0046] 2.4 Apcin administration

[0047] Apcin powder was dissolved in DMSO to a concentration of 80 mg / ml stock solution, which was stored in a -20°C refrigerator after being divided. Before each injection, an appropriate amount of stock solution was taken out and 40% PEG300 + 5% Tween80 + 50% ddH 2 O was diluted to a working solution (taking 1 mL of working solution as an example. Take 50 μL of 80 mg / mL clarified DMSO stock solution and add it to 400 μL PEG300, mix well to make it clear; add 50 μL Tween80 to the above system, mix well to make it clear; then continue to add 500 μL ddH 2 O was diluted to 1 mL. The working solution was prepared and used immediately). According to the dosage of 20 mg / kg body weight, an appropriate amount of working solution was injected intraperitoneally. The control group was given an equal amount of solvent (5% DMSO + 40% PEG300 + 5% Tween80 + 50% ddHO). 2 O) Intraperitoneal injection.

[0048] 2.5 Animal sampling

[0049] (1) Anesthesia: Each experimental mouse was intraperitoneally injected with 10 μL / g supersaturated tribromoethanol. After the anesthesia took effect, the mouse's chest was exposed and its limbs were fixed on the experimental board.

[0050] (2) Cardiac perfusion: Cut the mouse skin and sternum along the midline of the sternum to fully expose the chest cavity and heart. Make a micro incision at the right atrial appendage of the mouse, then quickly insert the syringe needle at the apex of the heart and slowly and evenly inject 20 mL of 0.01 M PBS within 5 minutes.

[0051] (3) Aorta collection: Cut the skin and subcutaneous membrane along the midline of the abdomen to expose the abdominal organs, and take the complete heart and aorta to the bifurcation of the sacroiliac arteries. After the complete aorta is taken out, the fat tissue around the blood vessels is peeled off under a stereo microscope (the dissection of the blood vessels with dissection should be gentle to avoid rupture of the blood vessels), and the ascending aorta segment of about 2 mm is retained. It is fixed with 4% paraformaldehyde for 4-6 hours and then transferred to 20% sucrose solution for storage. The remaining aortic tissue is immediately frozen in liquid nitrogen and stored at -80°C.

[0052] 2.6 Preparation of frozen tissue sections

[0053] (1) Fresh vascular tissue was collected and fixed in 4% paraformaldehyde for 4-6 hours.

[0054] (2) Transfer to 20% sucrose solution overnight.

[0055] (3) Transfer the cells to OCT for 1-2 min to reach equilibrium and allow the blood vessels to fill. Quickly freeze the cells in liquid nitrogen and store them in a -20°C refrigerator.

[0056] (4) After precooling with a Leica cryostat, the vascular tissue was sliced ​​into thin slices with a thickness of 5 μm and mounted on a glass slide.

[0057] 2.7EVG staining

[0058] That is the Verhoeff elastic fiber dyeing method, the specific operation is as follows:

[0059] (1) Place the frozen sections in a fume hood for 30 minutes to return to room temperature, place them in a 37°C incubator for 10-30 minutes, soak them in distilled water for 5 minutes, and use a histochemical pen to draw a circle around the tissue.

[0060] (2) Verhoeff staining solutions A, B, and C were mixed in a volume ratio of 5:2:2.

[0061] (3) Drip or immerse the frozen sections in Verhoeff stain for 10 min until the color turns dark black, then rinse with running water to remove excess stain.

[0062] (4) Differentiate with Verhoeff differentiation solution until the elastic fibers are clear (observed under a microscope for about 2 minutes and the elastic fibers are clear to the naked eye), and rinse with running water for 2-3 minutes.

[0063] (5) Treat with 95% ethanol for 1-3 seconds for rapid deiodination and rinse with tap water for 2-3 minutes.

[0064] (6) Drip or soak the sections with 5% sodium thiosulfate for 3 minutes and rinse with tap water for 2 minutes.

[0065] (7) Re-stain with VG staining solution for 40 seconds and rinse with distilled water for 2 minutes.

[0066] (8) Rapid differentiation was performed in 95% ethanol, followed by anhydrous ethanol for 2 × 2 min and xylene for 2 × 15 min.

[0067] (9) Add a drop of neutral gum and cover the sample with a glass slide. Place the sample in a well-ventilated place and observe under a microscope for analysis.

[0068] 2.8 Immunohistochemical staining

[0069] (1) Slice rewarming: Place frozen slices in a fume hood to return to room temperature for 30 minutes and soak in distilled water for 5 minutes.

[0070] (2) Fixation: Soak in 4% paraformaldehyde for 10 min, and then wash with PBS three times, each time for 3 min.

[0071] (3) Antigen repair: Prepare 1× sodium citrate antigen repair solution with distilled water. First, boil the repair solution in a microwave oven at medium heat for 5 minutes, and then place the slices in the repair solution and microwave at medium-low heat for 15 minutes.

[0072] (4) Cooling: After the slices were cooled naturally to room temperature, they were circled with a histological pen and then washed with PBS for 3 × 3 min.

[0073] (5) Membrane disruption: 0.1% TritonX-100, incubate at room temperature for 10 min, and wash with PBS for 3×3 min.

[0074] (6) Blocking: Add appropriate amount of endogenous peroxidase blocker, incubate at room temperature for 10 min, and wash with PBS for 3×3 min.

[0075] (7) Blocking: Add an appropriate amount of normal goat serum working solution for blocking and incubate at room temperature for 1 h;

[0076] (8) Incubation with primary antibody: Pour off the serum, add appropriate amount of primary antibody, and incubate at 4°C overnight;

[0077] (9) The sections were rewarmed at room temperature for 30 min, washed 3×3 min with PBS, and an appropriate amount of reaction enhancement solution was added. The sections were incubated at room temperature for 30 min, and washed 3×3 min with PBS.

[0078] (10) Incubation with secondary antibody: Add enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer, incubate at room temperature for 1 h, and wash with PBS 3×3 min.

[0079] (11) Color development: Add freshly prepared DAB color developing solution (A:B=1:50) dropwise, develop color according to the color development effect, and put into distilled water to stop the color development.

[0080] (12) Re-staining of cell nuclei: drip or dip the frozen sections with hematoxylin stain for 1 min, rinse with tap water to remove floating color. Add 1% hydrochloric acid alcohol differentiation solution for 5 s, rinse with tap water for 2 min to return to blue, and remove floating color.

[0081] (13) Downward dehydration: anhydrous ethanol 2 × 2 min, xylene 2 × 15 min.

[0082] (14) Add a drop of neutral gum and cover the sample with a glass slide. Place the sample in a well-ventilated place and observe under a microscope for analysis.

[0083] 2.9 Total RNA extraction and real-time fluorescence quantitative PCR

[0084] 2.9.1 Preparation before experiment

[0085] Reagents and consumables: isopropanol, chloroform, Trizol, DEPC water, anhydrous ethanol, 75% ethanol (prepared with anhydrous ethanol + DEPC water), RNA-specific tweezers, tissue scissors, RNA-free EP tubes, RNA-free pipette tips (1mL / 200μL / 10μL), RNA-free eight-row tubes / strips, RNA-specific grinding beads, biological sample homogenizer, cell scraper, grinding tubes.

[0086] 2.9.2 Total RNA extraction

[0087] (1) Transfer the EP tube containing the frozen aortic tissue into liquid nitrogen, and use tissue scissors to evenly chop the aorta. Transfer the chopped tissue into a grinding tube, add RNA grinding beads, add 1 mL of Trizol to the grinding tube in a fume hood, tighten the lid and evenly place it in a biological sample homogenizer for grinding until there are no obvious blocky tissues and the aorta becomes flocculent. Keep the whole operation on ice.

[0088] (2) Add 200 μL of chloroform to each tube, mix by inverting, shake vigorously for 1 min, let stand on ice for 15 min, wait for the liquid to separate, and then centrifuge at 4°C, 12,000 rpm for 15 min.

[0089] (3) Take the upper colorless aqueous phase and add it to a new EP tube. Add an equal volume of isopropanol, mix by inverting, and let stand at room temperature for 15 min.

[0090] (4) Centrifuge at 12,000 rpm for 15 min at 4°C. Observe whether there is a white precipitate. Remove the supernatant and add 1 mL of 75% ethanol to the EP tube for washing. Gently flick the precipitate off the bottom of the tube, but do not allow it to disperse.

[0091] (5) Discard the 75% ethanol, avoid contact with the white precipitate at the bottom, open the lid and ventilate in a clean bench until it becomes a translucent jelly.

[0092] (6) Depending on the amount of precipitation, add 5-50 μL of DEPC water, repeatedly pipette to dissolve the RNA, and centrifuge.

[0093] (7) The purity of the extracted RNA sample, including the A260 / 280 value and concentration, was measured using an ultra-micro spectrophotometer. The concentration was controlled at 300-500 ng / mL and the A260 / 280 value was 1.8-2.0.

[0094] 2.9.3 cDNA synthesis

[0095] (1) Removal of residual genomic DNA

[0096] Prepare the mixed solution according to Table 2 in an RNase-free centrifuge tube, mix gently by pipetting, and incubate at 42°C for 2 min.

[0097] Table 2 Residual genomic DNA removal reaction system

[0098]

[0099] (2) Reverse transcription reaction system configuration (20 μL system)

[0100] Directly add 4×HifairШSuperMix plus to the reaction tube in the first step and mix gently by pipetting.

[0101] Table 3 Reverse transcription reaction system

[0102]

[0103] The program settings are as follows:

[0104] Table 4 Reverse transcription reaction procedure

[0105]

[0106] 2.9.4 Real-time fluorescence quantitative PCR

[0107] The procedures were performed using the operating procedures of the Applied Biosystems 7500 Fast Real Time PCR System.

[0108] (1) Synthesize primers according to the primer sequences of the target genes to be detected (see Table 5), and dilute the primer stock solution according to the primer instructions.

[0109] Table 5 Real-time fluorescence quantitative PCR primer information

[0110]

[0111] (2) qPCR reaction system

[0112] The reaction system is shown in Table 6.

[0113] Table 6 qPCR reaction system

[0114]

[0115] Two-step PCR reaction procedure

[0116] The first step was pre-denaturation: 95°C for 30 s. The second step was PCR reaction: 40 cycles: 95°C for 3 s, 60°C for 20 s.

[0117] 2.10 Statistical analysis

[0118] The Shapiro-Wilk normality test was used. If the data met the normal distribution and the variance between the groups was equal, the Student's t-test was used to determine the significant difference between the two groups, and the results were presented in the form of mean ± standard deviation; if the above conditions were not met, the non-parametric Mann-Whitney U test was used, and the results were presented in the form of median. All data were statistically analyzed using GraphPad Prism V-7.0 software, and P < 0.05 was considered statistically significant.

[0119] 3. Experimental results

[0120] 3.1 Cell cycle regulatory protein CDC20 is abnormally elevated in human aortic dissection

[0121] By searching the human aortic dissection GEO database on the NCBI website, the dataset GSE52093 was screened out. This dataset included ascending aorta specimens including the dissection site removed by surgery from 7 Stanford type A dissection patients who underwent emergency surgery, and 5 healthy ascending aorta specimens from organ donors as dissection group controls. KEGG analysis of the aortic transcriptome data in this dataset revealed that cell cycle was one of the most significantly differential pathways in human aortic dissection lesions, with a total of 43 differentially expressed genes detected in this pathway ( Figure 1 A). A volcano plot analysis of all differentially expressed genes screened from this dataset showed that the five most significantly upregulated genes were CDC20, CHEK1, PTTG1, MCM4, and CDC7, and the five most significantly downregulated genes were CDKN1C, CDC14B, SKP1, CDKN1B, and RBL2. Among them, CDC20 expression was abnormally elevated, with the most significant change among all differentially expressed genes ( Figure 1 This finding is consistent with the conclusions stated in previous academic papers.

[0122] 3.2 CDC20 is also significantly increased in the mouse aortic dissection model

[0123] In order to clarify whether the increase of CDC20 is a concomitant phenomenon or a pathogenic factor in aortic dissection, we then verified it in a mouse aortic dissection model. First, by searching the mouse aortic dissection GEO database, we screened out the data set GSE116434, which contains the aortic transcriptome data of the mouse aortic dissection model constructed by BAPN combined with angiotensin II perfusion. The analysis results showed that the expression level of CDC20 in this model was significantly increased ( Figure 2In order to exclude the potential effect of angiotensin II on CDC20 expression, we further established a mouse aortic dissection model by simple BAPN induction. In this model, we used real-time fluorescence quantitative PCR technology ( Figure 2 B) and immunohistochemical staining ( Figure 2 Middle C) also confirmed that CDC20 expression was significantly increased. The above results indicate that abnormal increase of CDC20 may be a common pathological change in aortic dissection. In addition, whether it is a mouse aortic dissection model induced by BAPN alone or BAPN combined with angiotensin II, it can well simulate the pathological phenomenon of abnormal increase of CDC20 in human aortic dissection, which is suitable for subsequent pharmacodynamic research verification.

[0124] 3.3 CDC20-specific inhibitor Apcin significantly inhibits aortic dissection in mice

[0125] Next, the present invention administered Apcin, a specific inhibitor of CDC20, to a mouse aortic dissection model induced by BAPN combined with angiotensin II AngⅡ perfusion to observe the effect of Apcin on aortic dissection. The experiment was divided into a drug-treated group (Apcin group) and a solvent control group (Vehicle group). Each group induced aortic degeneration by adding BAPN (1g / kg / d) to drinking water. From the 21st day of BAPN feeding, angiotensin II (50μg / kg / d) was given subcutaneously for 3 consecutive days to increase vascular wall pressure to accelerate aortic lesions. After 28 days of BAPN feeding, the samples were collected; the Apcin group was given Apcin (20mg / kg) by intraperitoneal injection every other day from the start of BAPN feeding, and the Vehicle group was given an equal amount of solvent by intraperitoneal injection ( Figure 3 The results showed that among the 18 mice in the Vehicle group, a total of 8 mice (8 / 18) died of aortic dissection during BAPN feeding, while only 5 mice (5 / 16) died of aortic dissection among the 16 mice in the Apcin group ( Figure 3 B and E). The samples were collected and found that 15 mice (15 / 18) in the Vehicle group had visible aortic dissection lesions, while only 9 mice (9 / 16) in the Apcin group had visible aortic dissection lesions ( Figure 3 In addition, compared with the mice in the Vehicle group, the mice in the Apcin group not only had a lower incidence of aortic dissection ( Figure 3 D), the percentage of the segment with dissection in the total length of the aorta was also significantly reduced ( Figure 3 Middle F).

[0126] The statistical results of the maximum diameter of each aorta segment (ascending aorta segment, thoracic aorta segment and abdominal aorta segment) showed that compared with the Vehicle group, the maximum diameters of the ascending aorta segment and thoracic aorta segment in the Apcin group were significantly reduced, but there was no significant difference in the abdominal aorta segment ( Figure 3 Middle G). The above results prove that CDC20 inhibitor Apcin can significantly reduce aortic dissection lesions in mice.

[0127] 3.4 CDC20-specific inhibitor Apcin significantly inhibited BAPN-induced degenerative changes in the aortic media

[0128] Elastic fiber degradation, vascular smooth muscle loss, and extracellular matrix degradation are typical degenerative pathological changes of the media in aortic dissection. Among them, elastic fiber degradation can be semi-quantitatively scored by EVG staining, and the scoring criteria are: grade 1, no elastin degradation, elastic fibers are dense, regularly arranged, and wavy; grade 2, a small amount of elastin degradation, elastic fibers are loose and linear compared with grade I; grade 3, moderate elastin degradation, a small amount of elastic fiber rupture; grade 4, severe elastin degradation, elastic fibers are sparse, and multi-layer rupture occurs. Vascular smooth muscle loss can be quantitatively analyzed by α-SMA immunohistochemical staining, while extracellular matrix degradation is mainly mediated by matrix metalloproteinases MMP2 and MMP9, both of which can be quantitatively analyzed by immunohistochemical staining.

[0129] EVG staining results showed that the aortas of 18 mice in the Vehicle group had a score of 3-4 at a rate of about 15 / 18 (including 1 case of grade 3 and 14 cases of grade 4), and the score of 1-2 was only 3 / 18, with a median score of 4. In the Apcin group, the aortas of 16 mice had a score of 3-4 at a rate of 11 / 16 (including 5 cases of grade 3 and 6 cases of grade 4), and the score of 1-2 increased to 5 / 16, with a median score of 3 ( Figure 4 This suggests that Apcin inhibited CDC20 activity and significantly improved elastic fiber degradation in aortic dissection lesions in mice.

[0130] The results of α-SMA immunohistochemical staining showed that the α-SMA positive area in the vascular media of the Apcin group was significantly increased compared with that of the Vehicle group ( Figure 4 Middle B), indicating that Apcin can effectively inhibit the loss of vascular smooth muscle in aortic dissection lesions in mice after inhibiting CDC20 activity.

[0131] Finally, the results of MMP2 / MMP9 immunohistochemical staining showed that the positive areas of MMP2 and MMP9 in the aorta samples of the Apcin group were significantly reduced compared with those of the Vehicle group ( Figure 4(C and D), indicating that Apcin can significantly improve the degradation of extracellular matrix in mouse aortic dissection lesions after inhibiting CDC20 activity.

[0132] 3.5 CDC20-specific inhibitor Apcin significantly inhibits BAPN-induced aortic inflammatory cell infiltration

[0133] More and more studies have shown that abnormal activation of inflammation is an important pathogenic factor of aortic dissection, and inhibiting inflammation can reduce the progression of aortic dissection. Macrophages are the most abundant inflammatory cell type in aortic dissection lesions, and their main markers include CD68, F4 / 80, etc. CD68 immunohistochemical staining results showed that the CD68 positive area in the Apcin group was significantly lower than that in the Vehicle group ( Figure 5 A and B), suggesting that Apcin could significantly inhibit the infiltration of inflammatory cells in aortic dissection by inhibiting CDC20 activity.

[0134] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of CDC20 inhibitors in the preparation of drugs for preventing and / or treating aortic dissection.

2. The use according to claim 1, characterized in that The CDC20 inhibitor is Apcin.

3. The use according to claim 1 or 2, characterized in that: The aortic dissection is induced by 3-fumaric acid aminopropionitrile combined with angiotensin II.

4. The use according to claim 1, characterized in that The drug reduces the incidence and lesion area of ​​aortic dissection, improves vascular degenerative changes and inflammatory macrophage infiltration in aortic dissection lesions, and achieves the effect of preventing or treating aortic dissection.

5. A drug for preventing or treating aortic dissection, characterized in that: The medicine comprises a CDC20 inhibitor and pharmaceutically acceptable excipients.

6. The drug according to claim 5, characterized in that The CDC20 inhibitor is Apcin.

7. The drug according to claim 5, characterized in that The dosage forms of the drug include injection, powder, tablet, granule, capsule, solution, emulsion and suspension.

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