Application of indole-3-propionic acid in preparation of medicine for treating and / or preventing restenosis after vascular interventional operation

Abstract

The invention relates to the field of cardiovascular systems, discloses application of indole-3-propionic acid in preparation of a medicine for treating and / or preventing restenosis after vascular interventional operation, and provides a new thought and a theoretical basis for prevention and treatment of postoperative restenosis and research and development of targeted medicines.

Description

Technical Field

[0001] The present invention relates to the cardiovascular field, and in particular to an application of indole-3-propionic acid in preparing a medicine for treating and / or preventing restenosis after vascular intervention surgery. Background Art

[0002] Cardiovascular and cerebrovascular diseases have become the leading cause of death in China and developed countries. Vascular stenosis or occlusive lesions are important causes of death from cardiovascular diseases. Innovations in endovascular technology such as drug-coated balloons or eluting stents have achieved better therapeutic effects on vascular stenosis and occlusive lesions, but the incidence of restenosis is still as high as 17% in one year and 30%-40% in two years. Vascular restenosis caused by neointimal regeneration after endovascular treatment is still a serious clinical problem.

[0003] The human gut microbiota is essential for human health and is closely related to the pathogenesis of a variety of cardiovascular diseases. Increasing evidence from animal and human studies supports that the gut microbiota can affect the health and disease of the host, and that the gut microbiota can directly affect the development of cardiovascular disease through the production of metabolites such as bile acid, lipopolysaccharide, short-chain fatty acids, and trimethylamine-N-oxide. The gut microbiota produces a large number of metabolites, some of which are absorbed by the systemic circulation and have biological activity, while others are further metabolized by host enzymes and then affect the host as microorganisms. The gut microbiota acts as a virtual endocrine system, communicating with distal organs through metabolism-dependent pathways. Indole-3-propionic acid (IPA), a tryptophan metabolite of the gut microbiota, has been found to have anti-atherosclerotic and myocardial function-improving effects. At present, microbiota transplantation, microbiota remodeling, and exogenous supplementation of microbiota metabolites have become a hot topic for the treatment and prevention of cardiovascular diseases. However, there is no research and application of the gut microbiota in restenosis after cardiovascular intervention. Summary of the invention

[0004] The purpose of the present invention is to overcome the problem of the lack of application of intestinal flora in restenosis after cardiovascular intervention in the prior art, and to provide an application of indole-3-propionic acid in the preparation of a drug for treating and / or preventing restenosis after vascular intervention.

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides a use of indole-3-propionic acid in the preparation of a medicament for treating and / or preventing restenosis after vascular intervention surgery.

[0006] Preferably, the restenosis after vascular interventional surgery is caused by neointimal regeneration caused by mechanical damage during endovascular interventional surgery.

[0007] Preferably, the mechanical damage is caused by mechanical stretching.

[0008] Preferably, the indole-3-propionic acid inhibits the neointimal regeneration by reducing the proliferation and / or migration of vascular smooth muscle cells caused by the mechanical injury.

[0009] Preferably, reducing the proliferation and / or migration of vascular smooth muscle cells caused by the mechanical damage and thereby inhibiting the intimal neoplasia means that indole-3-propionic acid reduces the proportion of S phase cells in vascular smooth muscle cells and increases G0 / G1 and G2 / M phase arrest, thereby reducing the proliferation and migration of vascular smooth muscle cells and thereby inhibiting the intimal neoplasia of vascular smooth muscle cells.

[0010] A second aspect of the present invention provides a drug for treating and / or preventing restenosis after vascular intervention surgery, wherein the drug comprises indole-3-propionic acid.

[0011] Preferably, the medicament further comprises a pharmaceutically acceptable material.

[0012] Preferably, the drug comprises any one of an oral preparation, an injection and a surface coating agent, wherein the surface coating agent is coated on the surface of the stent and / or the balloon.

[0013] A third aspect of the present invention provides a use of indole-3-propionic acid in the preparation of a drug for reducing the expression of CDK2 in vascular smooth muscle cells.

[0014] A fourth aspect of the present invention provides an application in drug screening for treating and / or preventing restenosis after vascular intervention surgery.

[0015] Preferably, the application includes a method for screening a drug, wherein the screening method includes:

[0016] Treating vascular restenosis model animals or cells with the drug to be screened;

[0017] Detect the proliferation of vascular smooth muscle cells in the treated vascular restenosis model animals or cells;

[0018] Drug screening is performed based on the proliferation of the obtained vascular smooth muscle cells. If the drug to be screened reduces the proliferation of the vascular smooth muscle cells, the drug to be screened is the target drug obtained by screening.

[0019] The present invention establishes in vivo and in vitro models of vascular deformation injury and uses in vivo and in vitro experimental models to illustrate the role and specific mechanism of IPA in alleviating intimal regeneration after vascular mechanical stretch, providing new ideas and theoretical basis for the prevention and treatment of postoperative restenosis and the development of targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The establishment of in vivo and in vitro injury models, Figure 1 (A) is the femoral artery guidewire injury model. Figure 1 (B) is an in vitro vascular deformation injury model;

[0021] Figure 2 In vivo experiments have shown that IPA reduces intimal regeneration after mechanical stretching of blood vessels. Figure 2 (A) is a schematic diagram of the grouping and modeling of mice in each group. Figure 2 (B) Elisa method was used to detect the serum IPA level in mice given oral administration. Figure 2 (C) H&E method was used to detect the neointimal formation of femoral arteries in each group. Figure 2 (D) and Figure 2 (E) Statistical quantification diagram of the neointimal neoformation degree and intima / media ratio of the femoral arteries of each group of mice;

[0022] Figure 3 In vitro experiments have shown that IPA reduces the proliferation and migration of vascular smooth muscle cells after mechanical stretch. Figure 3 (A) The proliferation ability of cells in each group was detected by EdU method. Figure 3 (B) is the statistical quantification diagram of the EdU detection results. Figure 3 (C) CCK8 method was used to detect the proliferation ability of cells in each group. Figure 3 (D) and Figure 3 (E) The plate scratch and Transwell results of each group. Figure 3 (F) and Figure 3 (G) Statistical quantification diagram of the results of flat plate scratch test and Transwell experiment respectively;

[0023] Figure 4 In vitro experiments have shown that IPA reduces the expression of CDK2 in vascular smooth muscle cells. Figure 4 (A) is the PCR-Array results of the Ctrl group and the 10% MS group. Figure 4 (B) and Figure 4 (C) Western Blotting test results and statistical quantification graphs of each group. Figure 4 (D) is the RT-qPCR test results of each group. Figure 4 (E) and Figure 4 (F) Flow cytometry detection results and their statistical quantification graphs. DETAILED DESCRIPTION

[0024] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0025] 1. Materials and Methods

[0026] 1. Experimental Materials

[0027] 1.1 Experimental animals

[0028] The experimental animals required for this experiment were 8-week-old C57BL / 6J mice, all male mice were used as research subjects, and all mice were purchased from Chongqing Tengxin Biotechnology Co., Ltd. (SCXK (Yu) 2017-0007). All experimental mice were raised and related animal operations were carried out at the Experimental Animal Center of Southwest Medical University (License No.: SCXK (Chuan) 2013-17). Before animal modeling, all mice were fed freely and exposed to rhythmic day and night light at room temperature of 25°C. All aspects of mouse breeding and experimental processes were strictly carried out in accordance with the "Southwest Medical University Laboratory Animal Quality Management Measures", and this study was approved by the Animal Ethics Committee of Southwest Medical University.

[0029] 1.2 Experimental cells

[0030] The experimental cells required for this study were mouse aortic primary smooth muscle cells (MOVAS) isolated from the thoracic aorta of the above-mentioned experimental mice using tissue culture method under a stereomicroscope; the third generation of mouse primary aortic smooth muscle cells were used, and the smooth muscle cells were identified with primary cell identification-specific antibodies, and the nuclei were stained with DAPI to confirm that the cultured cells were smooth muscle cells for subsequent experiments.

[0031] 1.3 Main experimental reagents

[0032] The purchasing information of the main reagents used in this experiment is as follows. Others not mentioned are commercially available products.

[0033] DMEN / F 12 medium, purchased from Gibco, catalog number C11330500BT;

[0034] Fetal bovine serum, purchased from Gibco, catalog number A3160902;

[0035] Penicillin-streptomycin-amphotericin B mixed solution (100×), purchased from Solarbio, product number P1410-100ml;

[0036] Trypsin cell digestion solution (0.25%), purchased from BasalMedia, catalog number S310KJ;

[0037] Total RNA extraction kit, purchased from Foregene, catalog number RE-03111;

[0038] SYBR Green Realtime PCR Master mix, purchased from Toyobo, catalog number QPK-201;

[0039] ReverTra qPCR RT Master Mix, purchased from Toyobo, catalog number FSQ-301;

[0040] Gene primers were purchased from Sangon Company;

[0041] Rabbit anti-CDK2 monoclonal antibody, purchased from Abcam, catalog number ab32147;

[0042] Mouse anti-GAPDH monoclonal antibody, purchased from Proteintech, catalog number 60004-1-Ig;

[0043] Secondary antibody, HRP-conjugated Goat Anti-Mouse IgG (H+L), purchased from Proteintech, catalog number SA00001-1;

[0044] Secondary antibody, HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L), purchased from Proteintech, catalog number SA00001-2;

[0045] Specific antibodies for primary cell identification were purchased from abcam, catalog number: ab7817;

[0046] PAGE gel rapid preparation kit, purchased from Epizyme, catalog number PG112;

[0047] Three-color pre-stained protein Maker, purchased from Epizyme, catalog number WJ103L;

[0048] BCA protein detection kit, purchased from Beyotime, catalog number P0012;

[0049] ECL luminescent liquid, purchased from Epizyme, catalog number SQ203L;

[0050] Paraformaldehyde, purchased from Solarbio, catalog number P1110;

[0051] H&E staining kit, purchased from Solarbio, catalog number G1120;

[0052] RIPA lysis buffer (strong), purchased from Beyotime, catalog number P0013B;

[0053] 1M Tris-HCl, pH 6.8, purchased from Beyotime, catalog number ST768-1.0;

[0054] SDS-PAGE protein loading buffer (5X), purchased from Beyotime, catalog number P0285-2ml;

[0055] Glycine, purchased from BBI Life Sciences, catalog number A610236-0500;

[0056] Tris, purchased from BBI Life Sciences, catalog number A600194-0500;

[0057] Skim milk, purchased from BBI Life Sciences, catalog number A600669-0250;

[0058] Trizol, purchased from Invitrogen&trade, catalog number 10296010;

[0059] Chloroform, purchased from Sigma-Aldrich, product number C2432-25ML;

[0060] Cell cycle detection kit, purchased from Beyotime, catalog number C1052;

[0061] Indole-3-propionic acid (IPA), purchased from Sigma-Aldrich, product number 220027-10;

[0062] Crystal violet stain, purchased from Beyotime, catalog number C0121;

[0063] CCK8 reagent, purchased from Beyotime, catalog number C0038;

[0064] Transwell chamber, purchased from JETBIOFIL, catalog number TCS003024;

[0065] Mouse IPA Elisa detection kit, purchased from Mlbio, catalog number ml823265;

[0066] EdU proliferation detection kit was purchased from BBI Life Sciences, catalog number E607204-0050.

[0067] 2 Experimental methods

[0068] 2.1 Cultivation of primary mouse aortic smooth muscle cells

[0069] After the mice were anesthetized with 1% sodium pentobarbital (dose: 50 mg / kg), they were fixed in a supine position. The hair on the chest and abdomen of the mice was completely removed using a hair remover and a hair removal cream. After the skin was disinfected with iodine, the chest and abdominal cavities were opened in sequence using dissecting forceps and tissue forceps, the sternum was cut, and the internal organs were opened to one side to expose the heart. The right atrial appendage was cut open (a small amount of blood could be seen flowing out), a 10ml empty needle was inserted into the slightly upper position of the left apex, and 10ml PBS (phosphate buffer, PH=7.4) was perfused, and the aorta was repeatedly rinsed 2-3 times until the aorta was transparent. The intercostal arteries and aorta were bluntly freed along the mouse spine with micro forceps (the blood vessels could not be clamped, only the attached fat could be clamped), and the thoracic aorta was cut (above the renal plane) and immediately placed in a pre-cooled sterile PBS culture dish; under a stereo microscope, the fat tissue around the blood vessels was gently peeled off with micro forceps until the aorta became transparent and clean. Move to a new clean PBS dish, cut the aorta longitudinally with microscissors, lay the aorta flat, with the intercostal artery side facing outward and the inner side facing upward, use ophthalmic curved forceps to bluntly scrape off the endothelial cells, leaving the remaining media tissue thin and transparent. Wash the blood vessels repeatedly with saline to remove impurities such as fat tissue and blood clots around the blood vessels, then mix the media tissues of multiple blood vessels collected at one time and put them into a sterile PBS culture dish. Continue to press and fix with the left hand forceps, and use the right hand forceps to slightly lift and scrape upward. You can see that the media is tilted. Continue to scrape between the outer membrane and the media (scrape on the outer membrane, the media will naturally tilt), gradually move upward, press and scrape the media while moving upward, collect the media and immediately place it in sterile saline.

[0070] Use microscopic curved scissors to repeatedly cut the tissue blocks into 1×1mm size tissue blocks. Use a sterile Pasteur tube to evenly spread the cut tissue blocks in a T25 sterile culture bottle, with a spacing of 0.5cm between tissue blocks. Cover the bottle cap, stand the T25 culture bottle upright, and inject 7.5±2.5ml of DMEM / F12 culture medium into the bottle, then stand the culture bottle upright and place it in a 37℃, 5% CO2 incubator for 2h to allow the tissue blocks to completely dry and adhere closely to the bottle wall. Flip the culture bottle and lay it flat normally so that the tissue blocks are completely immersed in the culture solution. Continue to place the T25 culture bottle in the cell incubator for 7-8 days. During this period, move the culture bottle less frequently. After cells crawl out under the microscope, replace the new culture medium 3 days / time until subculture.

[0071] 2.2 Cell passaging

[0072] Observe the cell growth with a light microscope. When the cell density reaches more than 80% of the culture flask, perform subculture. The specific operation is as follows:

[0073] Use a sterile Pasteur tube to discard the old culture medium and add 5ml PBS to wash off the remaining culture medium and aging cells, add 2ml sterile 0.25% trypsin and place in a 37℃ incubator for digestion. After standing for 1min, move to an inverted microscope for inspection. When most cells are observed to shrink and float, add 4ml DMEM / F12 cell culture medium containing 10% fetal bovine serum (FBS) to terminate cell digestion. Use a sterile Pasteur tube to repeatedly blow the cells at the bottom of the bottle, transfer the resuspended cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm / min for 3 minutes, discard the old culture medium again after centrifugation, resuspend the cells with DMEM / F12 cell culture medium containing 10% fetal bovine serum, pass the cell suspension at 1:2-3, transfer to a 37℃, 5% CO2 incubator, and continue to maintain a suitable environment for cell growth.

[0074] 2.3 Cell cryopreservation

[0075] Select well-grown 2-6 generations of smooth muscle cells (MOVASs), remove the culture medium with a sterile Pasteur tube, add 2ml of 0.25% trypsin digestion solution to the bottle, place it in a 37°C, 5% CO2 incubator for 1min, observe the cells become round and wrinkled under an inverted microscope, and then add 4ml of DMEM / F12 medium containing 5% FBS to terminate trypsin digestion. Use a Pasteur tube to gently blow the bottom of the bottle, collect all the cell suspension and transfer it to a 15ml centrifuge tube, centrifuge at 1000rpm / min for 3min, remove the supernatant, prepare cell freezing solution according to the ratio of serum-free DMEM / F12 medium: serum: DMSO = 7:2:1, add 2ml of freezing solution, and dispense it into freezing tubes at 1ml / tube. First, place the cell freezing tubes in a gradient freezing box and put them in -80°C overnight, and put them in a liquid nitrogen tank for long-term storage the next day.

[0076] 2.4 Establishment of in vivo and in vitro models of vascular deformation injury

[0077] 2.4.1 Establishment of in vivo model of vascular deformation injury: The mouse femoral artery wire injury model (FAW) is used to simulate the vascular deformation injury associated with intravascular treatment.

[0078] After isoflurane inhalation anesthesia was stable, the mouse was fixed on a sterile animal operating table to keep the airway open. The Abbott CROSS-IT 200XT 0.014 size guide wire tip was selected. The working diameter of this guide wire tip was 0.25 mm, which was about 1.25-1.67 times the diameter of the normal mouse femoral artery (0.15-0.2 mm). When performing the mouse femoral artery guide wire deformation injury surgery, the mouse superficial femoral artery was freed under a stereomicroscope, and the deep femoral artery was protected. The superficial femoral artery was pre-punctured with a curved fine needle to assist the guide wire to enter the blood vessel through the puncture point. The guide wire stayed in the femoral artery for about 90-120 seconds, causing a short-term continuous vascular deformation stretch injury to avoid the guide wire from repeatedly advancing and retreating to damage the vascular endothelial cells. The proximal and distal ends of the puncture point were then ligated. The sham operation group only underwent skin incision and vascular freeing, and the modeling was completed. The modeling process was as follows Figure 1 (A) After 2 weeks, the mice were killed by cervical dislocation and the blood vessels near the puncture point were obtained.

[0079] 2.4.2 Establishment of an in vitro model of vascular deformation injury: Elastic chamber C802 system

[0080] like Figure 1 As shown in (B), the present invention proposes and establishes an elastic chamber C802 system, an in vitro cell experimental model for simulating vascular deformation damage. The elastic dish provided by the system can cause the adherent cells to produce continuous deformation of varying degrees for a certain period of time under the action of external force, and the vascular deformation damage caused by intravascular treatment is simulated by this system. The elastic dish is an elastic silicone dish-shaped device, which can be formed into a uniform deformation stretch by a matching stretching ruler. In the preliminary experiment, 5%, 10%, and 20% deformation stretches were used, and the deformation was maintained for 2 minutes. It was observed that the 10% deformation stretch had little effect on the subsequent culture state of the cells, and the 10% deformation stretch was in line with the actual situation of intravascular treatment surgery. Subsequent experiments were carried out with this degree of deformation.

[0081] 2.4.3 Frozen sections

[0082] Take out the tissue (from 2.4.1 and 2.4.2) embedded with OCT embedding agent (Optimal cutting temperature compound) from the -80℃ freezer, put it into the freezing microtome, and slice it into 10μm slices after balancing for 30 minutes. Flatten the slices with a brush, adhere the tissue slices with adhesive slides, and store them in a -80℃ freezer.

[0083] 2.5 RNA purification and reverse transcription experimental methods

[0084] 2.5.1 Total RNA extraction

[0085] (1) Different approaches are adopted for cells and blood vessels, as follows:

[0086] ① Cells: discard the cell culture medium, wash with sterile PBS three times, add 1 ml Trizol to the culture dish, place on ice for 5 min, repeatedly blow the cells, and collect them into a sterile enzyme-free 1.5 ml EP tube;

[0087] ② Blood vessels: Place the vascular tissue in a sterile, enzyme-free 1.5 ml EP tube, add 4 3 mm grinding steel beads, place the EP tube in a pre-cooled tissue grinder, grind at 60 Hz for 30 seconds, add 1 ml Trizol to the obtained vascular powder, lyse on ice for 30 minutes, and collect into a sterile, enzyme-free 1.5 ml EP tube.

[0088] (2) Add 200 μl of chloroform to the treated sample, mix thoroughly by inversion, and place on ice for 5 minutes;

[0089] (3) Centrifugation at 4°C and 13,000 rpm for 15 min;

[0090] (4) Gently pipette 400 μl of the upper aqueous phase into a new 1.5 ml EP tube, add an equal volume of isopropanol, mix well, and centrifuge at 4°C and 13,000 rpm / min for 10 min;

[0091] (5) Discard the supernatant and wash the RNA precipitate with 1 ml of 70% ethanol, 1 ml of 80% ethanol, 1 ml of 90% ethanol, and 1 ml of 100% ethanol, respectively. The ethanol solutions of different concentrations were all prepared with DEPC water.

[0092] (6) After washing, discard the supernatant and place the tube on an enzyme-free workbench to dry the RNA precipitate in the EP tube. Then add 20 μL of enzyme-free water to each 1.5 ml enzyme-free EP tube.

[0093] (7) After dissolving the RNA precipitate, the RNA concentration and purity were measured using a NanoDrop2000 / 2000c instrument and stored at -80°C for a long time.

[0094] 2.5.2 Reverse transcription

[0095] On ice, prepare the extracted RNA according to the reverse transcription reaction system (see Table 1), and perform the reaction at the following temperatures: heating to 37°C for 15 min; reverse transcription reaction at 55°C for 5 min; enzyme inactivation reaction at 98°C for 5 min; at 4°C, perform cDNA amplification or store at -20°C after the reaction.

[0096] Table 1Reverse transcription reaction system

[0097]

[0098] 2.5.3 Primer design

[0099] According to the β-actin and CDK2 gene sequences in NCBI, the primer sequences were designed using the NCBI online Primer designing tool (see Table 2).

[0100] Table 2Primers sequence for qRCR

[0101]

[0102] 2.6 RT-qPCR reaction

[0103] 2.6.1 Prepare the RT-qPCR reaction system as shown in Table 3.

[0104] Table 3 RT-qPCR reaction system

[0105]

[0106] 2.6.2 Sample addition

[0107] In a sterile and enzyme-free environment, place a dedicated 96-well plate on ice and add the reaction reagents according to Table 3. After the addition of the sample, centrifuge it in a dark place and place it in a fluorescent quantitative PCR instrument for RT-qPCR reaction. -△△ct The relative expression levels of related genes were calculated by the method, and the Ct value was the number of cycles experienced by the fluorescence signal in each reaction tube to reach the set threshold.

[0108] 2.7 Western blotting

[0109] 2.7.1 Preparation of protein samples

[0110] 2.7.1.1 Cell samples

[0111] (1) Take the cell sample, discard the original culture medium in the cell culture dish, and wash it with PBS buffer three times;

[0112] (2) Add 150 μl of protein lysis buffer (lysis buffer ratio is RIPA lysis buffer: PMSF = 100:1) to the culture dish and lyse on ice for 30 min;

[0113] (3) Use a clean cell scraper to scrape the lysed cells and transfer them to the corresponding 1.5 ml centrifuge tube;

[0114] (4) Place the centrifuge tube containing the protein suspension lysate in a high-speed centrifuge precooled at 4°C and centrifuge at 14,000 rcf for 15 minutes.

[0115] 2.7.1.2 Tissue samples

[0116] (1) Wash the tissue sample to be tested with PBS buffer to remove residual blood clots or culture medium;

[0117] (2) Transfer the tissue to a 1.5 ml centrifuge tube. The length of vascular tissue in each centrifuge tube should not exceed 5 mm.

[0118] (3) Add 200 μl of protein lysis buffer (lysis buffer ratio is RIPA lysis buffer: PMSF = 100:1) to each centrifuge tube;

[0119] (4) Place 2-3 steel balls into each centrifuge tube;

[0120] (5) Place the centrifuge tube in a tissue grinder precooled to 4°C and grind it into pieces. Set the grinder to 60 Hz for 30 seconds each time and repeat 5 times.

[0121] (6) After grinding, place the centrifuge tube on ice for 30 minutes;

[0122] (7) After lysis, transfer the suspension to a 1.5 ml centrifuge tube and place the centrifuge tube containing the protein suspension lysate in a high-speed centrifuge precooled at 4°C, set to 14000 rcf, and centrifuge for 15 min.

[0123] 2.7.2 Determination of protein concentration by BCA method

[0124] (1) After centrifugation, transfer the supernatant of the protein sample prepared in step 2.7.1 to a new 1.5 ml centrifuge tube and mark it;

[0125] (2) Calculating the measured absorbance value of the standard sample with the known concentration of the standard sample by the BCA method to obtain a standard curve, and then substituting the absorbance value into the standard curve to obtain the concentration of the original protein sample;

[0126] (3) Add 5× loading buffer to the protein sample at a ratio of 1:4, mix thoroughly, heat and denature it in a 100°C metal bath for 10 min, and then store the protein sample in a -20°C low-temperature refrigerator.

[0127] 2.7.3 Glue making

[0128] (1) Wash the long and short glass. When installing, the left and right sides and the bottom edges of the long and short glass must be aligned. Clamp and fix them to prevent leakage. Then place them on the glue rack and pad the sponge strips underneath to prevent leakage. There are three thickness specifications of the glue board: 0.75m, 1mm, and 1.5mm;

[0129] (2) According to the thickness of the rubber sheet, take equal volumes of lower layer glue solution and lower layer glue buffer solution in a container and mix them evenly. For 0.75 mm rubber sheet, take 2.0 ml of each of the two solutions, for 1 mm rubber sheet, take 2.7 ml of each of the two solutions, and for 1.5 mm rubber sheet, take 4.0 ml of each of the two solutions. Then, add a certain amount of improved coagulant to each of them and mix them evenly. For 0.75 mm rubber sheet, take 40 ul of the improved agent, for 1 mm rubber sheet, take 60 ul of the improved agent, and for 1.5 mm rubber sheet, take 80 ul of the improved agent.

[0130] (3) Pour the obtained mixed solution into the glass plate so that the distance between the liquid surface and the upper edge of the short glass plate is 1-2 mm longer than the tooth comb;

[0131] (4) Gluing: Take an appropriate amount of anhydrous ethanol (or isopropyl alcohol or water) and inject it into the glue plate;

[0132] (5) After the lower layer of glue solidifies, pour out the pressure line solution and absorb the solution with absorbent paper. When there is a refraction line between the isopropyl alcohol and the glue, it means that the glue has solidified, which takes about 5-10 minutes.

[0133] (6) After shaking the upper gel and upper gel buffer, take equal volumes of upper gel and upper gel buffer, 0.5 ml of each buffer for 0.75 m gel plate, 0.75 ml of each buffer for 1 m gel plate, and 1.0 ml of each buffer for 1.5 m gel plate, and mix well; add 10 ul, 15 ul, and 20 ul of the improved coagulant to the three different specifications of gel plates, respectively, mix well, inject the above upper gel mixture into the gel plate, and insert the tooth comb;

[0134] (7) Wait for the upper layer of gel to solidify (about 10-15 minutes), place the gel rack into the electrophoresis tank, fill it with electrophoresis solution, and remove the comb.

[0135] 2.7.4 Sample loading and electrophoresis

[0136] (1) Sample loading: add markers on both sides, add 5ul to the leftmost sample well, and add 1ul to the rightmost sample well;

[0137] (2) Concentrating gel: constant voltage 80mV, electrophoresis time is determined by the bromophenol blue indicator reaching the green area;

[0138] (3) Separation gel: Constant voltage 120mV, terminate electrophoresis when bromophenol blue reaches the bottom edge of the electrophoresis gel.

[0139] 2.7.5 Transfer

[0140] (1) Precool 1X transfer buffer at 4°C, place the cut PVDF / NC membrane in the transfer buffer, and place the sponge, filter paper, and transfer plate in the precooled transfer buffer;

[0141] (2) Use a gel cutting board to gently cut the gel sheet in the direction of the tooth comb after electrophoresis, then use the gel cutting board to cut off the excess gel, and then place it in the pre-cooled transfer solution to pre-cool for 5 minutes;

[0142] (3) A transfer sandwich is made in the order of "black transfer plate - one or two layers of sponge - filter paper - glue - PVDF / NC membrane - filter paper - sponge - transparent transfer plate", wherein the smooth surface of the filter paper between the sponge and the glue faces the glue, and the rough surface faces the sponge. The glue refers to the glue after electrophoresis. After all bubbles are removed, it is fixed on the red transfer rack and placed in the electrophoresis tank. The electrophoresis tank is pre-filled with transfer solution (about 1300 ml). The black negative electrode and the black transfer plate are in the same direction. The entire transfer device is placed in an ice box to cool down;

[0143] (4) Constant current 400 mA, calculate the time according to the protein molecular weight displayed on the membrane during WB operation. The transfer time is calculated as the molecular weight of the target protein + 5 min. Generally speaking, the molecular weight of common proteins is between 20-80, and the transfer time does not exceed 2 h.

[0144] 2.7.6 Closure

[0145] (1) After the transfer is completed, remove the transfer sandwich, with the black side facing the palm of your hand, turn over the other side of the transfer plate, and check whether the transfer is complete by observing whether the high molecular weight marker is transferred to the membrane. If not, do not move the sandwich contents, close the transfer plate and continue the transfer for a while. If the transfer is complete, proceed to the next step. Before blocking, remove the membrane and wash it three times with TBST, 5 minutes each time;

[0146] (2) Preparation of blocking solution: Add 5 g skim milk powder per 100 ml TBST, mix well, remove the precipitate or filter;

[0147] (3) Block at room temperature for 1 hour or shake at 4°C overnight;

[0148] (4) After blocking, wash with TBST three times, 5 minutes each time.

[0149] 2.7.7 Incubation of primary and secondary antibodies

[0150] The primary antibody (CDK2 monoclonal antibody or GAPDH monoclonal antibody, GAPDH monoclonal antibody as internal reference) was diluted according to the antibody instructions and incubated with the membrane on a shaker at 4°C overnight (about 12-15h). After the incubation, the membrane was washed 3 times with TBST, each time for 5min, and then incubated with the secondary antibody (diluted according to the antibody instructions) and incubated at room temperature for 2h.

[0151] 2.7.8 Development

[0152] Prepare the developer: Add ECL developer kit (ECL.chemiluminescence kit) A:B solution = 1:1 into a light-proof container and prepare it for immediate use. One strip requires 120ul. After development, use Image J to analyze the grayscale value and compare the expression levels.

[0153] 2.8 Cell cycle detection

[0154] 2.8.1 Preparation of cell samples

[0155] The cells were digested with trypsin cell digestion solution to make the cells detach from the wall, and the smooth muscle cells of the 3rd to 6th generations were collected into a centrifuge tube and centrifuged at 1000g for 5 minutes. After centrifugation, the supernatant was removed, and about 1 ml of 4°C pre-cooled PBS buffer (the PBS used in the present invention is 0.01M PBS buffer (PH 7.2-7.4)) was added to resuspend the cells, and the cells were centrifuged at 1000g again, the supernatant was removed, and the bottom of the centrifuge tube was gently tapped to disperse the cells to prevent the cells from clumping.

[0156] 2.8.2 Cell fixation

[0157] Add 1 ml of 4°C pre-cooled 70% ethanol to the centrifuge tube, gently pipette to mix, and fix at 4°C for 2 hours. After fixation, centrifuge at 1000g for 5 minutes, remove the supernatant after centrifugation, add about 1 ml of 4°C pre-cooled PBS, resuspend the cells, centrifuge the cells at 1000g again, and remove the supernatant.

[0158] 2.8.3 Cell staining

[0159] Refer to Table 4 to prepare propidium iodide staining solution.

[0160] Table 4 Staining solution configuration

[0161]

[0162] Add 0.5 ml of the prepared staining solution in Table 4 to each tube of cell sample, slowly and fully resuspend the cells, incubate at 37°C in the dark for 30 minutes, and detect by flow cytometer within 24 hours.

[0163] 2.9CCK-8 proliferation assay

[0164] Take 3-6 generations of mouse aortic smooth muscle cells with good growth, add DMEM / F12 medium containing 500 μM IPA for 2 hours in advance, then perform 10% deformation injury, and then digest with trypsin to prepare cell suspension, and mix thoroughly;

[0165] Count the cell suspension, adjust the cell concentration to be consistent, add 100 μl of cell suspension to each well of the 96-well cell culture plate, and repeat 3 times for the same sample. After culturing in a 37°C incubator for 24 hours, add 10 μl of CCK-8 solution to each well, and place the 96-well plate in the incubator for 1 hour.

[0166] The 96-well plate was taken out and placed in an ELISA reader for analysis. The absorbance value (OD) at 450 nm was measured by the ELISA reader, and the cell survival rate was calculated by the formula as follows:

[0167] Cell survival rate = (experimental group OD-blank group OD) / (control group OD-blank group OD) × 100%, wherein the blank group is DMEM / F12 culture medium, used to eliminate the absorbance deviation caused by the color of the culture medium, the control group is cells without any treatment (corresponding to the Ctrl group of the present invention), and the others are experimental groups (corresponding to the 10% MS group, 10% MS+500μM IPA group, and IPA 500μM group in subsequent experiments of the present invention).

[0168] 2.10 Transwell assay

[0169] 2.10.1 Cell sample preparation

[0170] Take 3-6 generations of mouse aortic smooth muscle cells with good growth, add DMEM / F12 medium containing 500μM IPA for 2 hours in advance, then perform 10% deformation injury, trypsin digestion, resuspend in serum-free medium and count, take an appropriate amount of cell suspension and dilute it to 1.6*10^4 cells / ml;

[0171] The Transwell chamber was removed from the 24-well plate and placed in a new 24-well plate. The diluted cells were added into the Transwell chamber, 300ul / chamber (density 5000 cells / well);

[0172] Add 700 μl / well of F12 / DMEM medium containing 10% serum to an empty 24-well plate, then gently place the Transwell chamber containing the diluted cells into the 24-well plate containing F12 / DMEM medium and culture for 24 hours.

[0173] 2.10.2 Cell fixation

[0174] After 24 hours, take a new 24-well plate, add 1 ml of 4% paraformaldehyde to each well according to the order of samples, take out the 24-well plate wells of the Transwell chamber after 24 hours of culture, take out the chamber, pour out the culture medium, and then use tweezers to place the chamber into the 24-well plate filled with 4% paraformaldehyde for fixation at room temperature for 30 minutes.

[0175] 2.10.3 Dyeing

[0176] Fill the empty 24-well plate with an appropriate amount of pure water, gently place the fixed chamber into the well plate filled with pure water, wash three times, 5 minutes each time, then add 1 ml of crystal violet (enough to immerse the bottom of the transwell chamber), gently place the washed chamber into the crystal violet, and incubate at room temperature for 20 minutes.

[0177] 2.10.4 Cleaning and Photography

[0178] After staining, wash the chamber three times according to the method in 2.10.3, each time for 5 minutes until the water in the chamber becomes clear;

[0179] Turn the cleaned chamber upside down on kitchen paper to remove excess water, and use a cotton swab to gently wipe the cells on the inner wall of the chamber, leaving only the cells on the outer wall of the chamber; turn the processed chamber upside down on kitchen paper to continue draining the water, and take pictures with an optical microscope after 24 hours.

[0180] 2.11 Flat plate scratch test

[0181] Take the 3rd to 6th generation mouse aortic smooth muscle cells with good growth, add DMEM / F12 medium containing 500 μM IPA for 2 hours in advance, and then perform 10% deformation injury for 2 minutes, add trypsin to digest the cells, and adjust the cell number to 6000 / well using a cell counting plate;

[0182] Take 2 ml of each group of cell suspension and plate it in a six-well plate, then place it in a 37°C incubator containing 5% CO2 to incubate the cells. When the cell density grows to 80%-90%, use a sterile 200 μl pipette tip to make a cross scratch perpendicular to the well plate, keeping the pipette tip vertical.

[0183] Wash the cells with PBS 3-4 times, add culture medium, collect the picture at this time as the 0h control, and continue to culture the cells in a 37℃, 5% CO2 incubator;

[0184] An inverted microscope was used to record the healing of the wound for 48 hours, and 3-5 fields of view were selected for photography. The scratch area was measured using ImageJ, and the cell migration rate was analyzed.

[0185] 2.12 EdU experiment

[0186] ① Take 3-6 generations of primary mouse aortic smooth muscle cells (MOVAS) with good growth, add DMEM / F12 medium containing IPA500μM for 2 hours in advance, then perform 10% deformation injury for 2 minutes, and culture in an incubator overnight;

[0187] ② Dilute the EdU solution with DMEM / F12 medium containing 10% FBS at a ratio of 1:1000 to obtain 50μM EdU medium, add 500μl EdU solution to step ① and continue incubating in the cell culture incubator for 2h;

[0188] ③Discard the culture medium, wash the cells with PBS for 3 times, 5 minutes each time, then add 1 ml of 4% paraformaldehyde to fix at room temperature for 30 minutes. After fixation, discard the fixative, wash the cells with PBS for 3 times, 5 minutes each time, then add PBS solution containing 2 mg / mL glycine and incubate at room temperature for 5 minutes. After incubation, add PBS solution containing 0.5% TritonX-100 and incubate at room temperature for 10 minutes, then wash the cells with PBS for 3 times, 5 minutes each time.

[0189] ④ After washing, add 500 μl of 1×TAMRA red fluorescent solution and incubate at room temperature in the dark for 30 min. After incubation, wash the cells 3 times with PBS, 5 min each time;

[0190] ⑤ Add 500 μl Hoechst 33342 solution and incubate at room temperature for 30 min. After incubation, wash the cells 3 times with PBS, each time for 5 min, and observe under a fluorescence microscope and take pictures.

[0191] 2.13 H&E staining

[0192] Take out the frozen sections from 2.4.3 and rewarm them at room temperature. After rewarming, stain with hematoxylin solution for 2 minutes, wash with distilled water for 3 times, 5 minutes each time, differentiate with differentiation solution for 10 seconds, wash with distilled water for 3 times, 5 minutes each time, and then stain with eosin solution for 20 seconds. After staining, quickly dehydrate with 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol, each for 2 seconds. After dehydration, slices were immersed in 100% ethanol for 1 minute, transparentized with xylene twice, 1 minute each time, and sealed with neutral gum.

[0193] 2. Experimental Results

[0194] 1. In vivo experiments have shown that IPA reduces intimal regeneration after mechanical stretching of blood vessels

[0195] C57BL / 6J mice of 8 weeks old and weighing 20-25 g were selected and grouped as follows:

[0196] ①C57BL / 6J control mouse group, without any treatment, recorded as Ctrl group;

[0197] ②C57BL / 6J mice group, gavaged with PBS buffer and injured the femoral artery with a guidewire, recorded as PBS+FAW group;

[0198] ③C57BL / 6J mouse group, intragastrically administered with IPA (20 mg / kg), and femoral artery guidewire injury was performed, recorded as IPA 20 mg / kg + FAW group;

[0199] ④C57BL / 6J mouse group, intragastrically administered with IPA (40 mg / kg), and concurrently performed femoral artery guidewire injury, recorded as IPA 40 mg / kg+FAW group.

[0200] Among them, ① is the control group, ② is the PBS+surgery group, ③ and ④ are the IPA+surgery groups. According to the above grouping, the mice were gavaged with the corresponding reagents and doses according to their weight. The PBS+surgery group was gavaged with an equal amount of PBS buffer for 10 consecutive days. After 10 days, the mice in the corresponding groups underwent femoral artery guidewire injury surgery. 14 days after surgery, samples were taken for subsequent experiments. This method can be used to suggest an animal model with high serum IPA. Mice modeled as described above were taken, and the proximal blood vessels of the puncture point were sampled 14 days after surgery. The blood vessels of each group were stained with hematoxylin-eosin (H&E) to observe the degree of intimal neoplasia in each group, confirming that IPA after vascular deformation injury reduced intimal neoplasia after mechanical stretching of the blood vessels. The results are as follows Figure 2 shown.

[0201] in, Figure 2 (A) Schematic diagram of the grouping and modeling of mice in each group;

[0202] Figure 2 (B) The serum IPA levels of mice in each group were detected by Elisa method. The results showed that the serum IPA levels of mice gavaged with 20 mg / kg and 40 mg / kg IPA were significantly increased;

[0203] Figure 2 (C) The H&E method was used to detect the neointimal formation of the femoral arteries of mice in each group. It can be seen that the neointimal formation of mice gavaged with IPA was significantly lower than that of the group gavaged with PBS, and it was concentration-dependent. Figure 2 (D) and 2(E) are the statistical quantification diagrams of the degree of femoral artery neointimal neoplasia and the intima / media ratio of each group of mice, respectively. It can be seen that the degree of femoral artery neointimal neoplasia in the PBS+FAW group was higher than that in the two groups of mice gavaged with IPA, and the degree of neointimal neoplasia in the IPA 40 mg / kg+FAW group was lower than that in the IPA 20 mg / kg+FAW group, indicating that IPA improved the neointimal neoplasia caused by femoral artery guidewire injury in a concentration-dependent manner.

[0204] 2. In vitro experiments have shown that IPA reduces intimal regeneration after mechanical stretching of blood vessels

[0205] Primary mouse aortic vascular smooth muscle cells (MOVAS) were pretreated with IPA at different concentration gradients (16000 μM-125 μM) to detect the safe concentration of IPA and obtain the IC value of IPA for MOVAS. 50 The concentration of 500 μM was 3500 μM, so the concentration that was effective and safe for MOVAS was selected for subsequent experiments. The cells in subsequent experiments were from MOVAS and grouped as follows:

[0206] ①Ctrl group, no processing;

[0207] ②10% MS group, 10% degree of mechanical stretch injury;

[0208] ③10% MS+500μM IPA group: MOVAS were treated with 500μM IPA for 2h, and then subjected to 10% mechanical stretch injury using the elastic chamber C802 system;

[0209] ④IPA 500μM group: MOVAS were treated with 500μM IPA for 2h.

[0210] After 24 hours, the proliferation ability of MOVAS in each group was detected by EdU and CCK8 methods, and the migration ability of MOVAS was detected by flat scratch and Transwell methods. The results are shown in Figure 3 shown.

[0211] Figure 3 (A) The proliferation ability of each group of cells was detected by EdU method, where the first row is the blue fluorescence image of the cell nucleus of each group of cells, the second row is the red fluorescence image of the proliferating cells labeled with EdU, and the third row is the co-localization image of the cell nucleus and EdU. The first column is the cells of the Ctrl group, the second column is the cells of the 10% MS group, the third column is the cells of the 10% MS+500μM IPA group, and the fourth column is the cells of the IPA 500μM group. It can be seen that the proliferation ability of smooth muscle cells increased significantly after 10% mechanical stretch, and IPA treatment can reduce the proliferation of smooth muscle cells caused by 10% mechanical stretch;

[0212] Figure 3 (B) is a statistical quantification diagram of EdU detection. It can be seen that 10% mechanical stretch can significantly increase the proliferation ability of smooth muscle cells, but IPA treatment can improve this phenomenon and inhibit the proliferation of smooth muscle cells caused by mechanical stretch.

[0213] Figure 3(C) The proliferation ability of cells in each group was detected by CCK8 method. It can be seen that the proliferation ability of smooth muscle cells in the 10% MS deformation group increased, but this result could be reversed after further IPA treatment with the same deformation;

[0214] Figure 3 (D) and Figure 3 (E) The plate scratch and Transwell results of each group. Figure 3 (D) The first and second rows are the results of 0 h and 48 h, respectively, and the first to fourth columns are the results of the Ctrl group, 10% MS group, 10% MS+500μM IPA, and IPA 500μM group, respectively; Figure 3 (E) The first picture in the first row shows the migration results of Ctrl cells, the second picture in the first row shows the migration results of 10% MS group cells, the first picture in the second row shows the migration results of 10% MS+500μM IPA group cells, and the second picture in the second row shows the migration results of IPA 500μM group cells. It can be seen that the migration ability of smooth muscle cells increased after 10% mechanical stretch, but this result can be reversed after IPA treatment;

[0215] Figure 3 (F) and Figure 3 (G) are statistical quantification diagrams of the results of the flat scratch test and the Transwell test. It can be seen that 10% mechanical stretch can significantly improve the migration ability of smooth muscle cells, but IPA treatment inhibits the migration of smooth muscle cells induced by mechanical stretch.

[0216] 3. IPA inhibits intimal neoplasia by reducing the increased expression of CDK2 (Cyclin-Dependent Kinase 2) in vascular smooth muscle cells caused by mechanical stretch.

[0217] MOVAS cells were grouped as follows:

[0218] ① Control group, cells were not treated in any way;

[0219] ②10% MS deformation group, cells underwent 10% mechanical stretch injury;

[0220] ③10% deformation + IPA treatment group, cells were treated with 500 μM for 2 h, then subjected to 10% mechanical stretch injury, and then treated with 500 μM for 24 h;

[0221] ④IPA treatment group, cells were treated with 500 μM.

[0222] 3.1 The PCR-Array method was used to detect the difference in gene expression between the control group and the 10% MS deformation group. The results are as follows Figure 4As shown in (A), con corresponds to the Ctrl group, and p10 corresponds to the 10% MS deformation group. It was found that the expression of CDK2 in the 10% MS deformation group was significantly higher than that in the Ctrl group.

[0223] 3.2 Western blotting was used to detect the expression differences of CDK2 in transcription and translation in each group of cells. The results are as follows Figure 4 As shown in (B), the first row shows the expression level of CDK2 protein in each group of cells, and the second row shows the expression level of reference protein in each group of cells.

[0224] In the first row, immunoblotting showed that the expression of CDK2 protein in the 10% MS deformation group was significantly higher than that in the Ctrl group, but the expression of CDK2 protein in the 10% deformation + IPA treatment group was significantly lower than that in the 10% MS deformation group, indicating that IPA can inhibit the increase in CDK2 expression in cells caused by 10% MS deformation. Figure 4 As shown in (C), the difference is statistically significant.

[0225] 3.3 Total RNA was extracted from cells in each group and RT-qPCR was performed to detect CDK2 mRNA levels. The results are as follows Figure 4 As shown in (D), it can be seen that the expression of CDK2 mRNA in the cells of the 10% MS deformation group was significantly increased compared with the control group, while the expression of CDK2 mRNA in the 10% MS deformation + IPA500μM treatment group was significantly decreased compared with the 10% MS deformation group, and the difference was statistically significant.

[0226] 3.4 Flow cytometry was used to detect the cell cycle changes in the above groups. The results are as follows Figure 4 As shown in (E), Figure 4 (E) The first two figures are the cell cycle changes of the Ctrl group and the 10% MS group, Figure 4 (E) The second row of two figures are the cell cycle changes of the 10% MS deformation + IPA500μM treatment group and the IPA treatment group. It can be seen that compared with the Ctrl group, the proportion of S phase (corresponding to S-Phase in the figure) in the 10% MS deformation group was significantly increased, and the G0 / G1 phase (corresponding to G0G1 in the figure) and G2 / M phase (corresponding to G2M in the figure) arrests were reduced, while the proportion of S phase in the 10% MS deformation + IPA500μM treatment group was significantly reduced compared with the 10% MS deformation group, and the G0 / G1 and G2 / M phase arrests increased. The statistical results are as follows Figure 4 As shown in (F), the difference is statistically significant.

[0227] It can be seen that 10% deformation treatment significantly increased the S phase level of cells, thereby increasing the cell proliferation and migration ability, and further adding IPA treatment to cells with this deformation can reverse the increase in the S phase level of smooth muscle cells in the cell cycle caused by deformation damage.

[0228] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. Use of indole-3-propionic acid in the preparation of a drug for treating and / or preventing restenosis after vascular intervention surgery.

2. The use according to claim 1, wherein: The restenosis after vascular intervention surgery is caused by neointimal formation caused by mechanical damage during endovascular intervention surgery.

3. The use according to claim 2, wherein: The mechanical damage is caused by mechanical stretching.

4. The use according to claim 2 or 3, wherein: The indole-3-propionic acid inhibits the neointimal regeneration by reducing the proliferation and / or migration of vascular smooth muscle cells caused by the mechanical injury.

5. The use according to claim 4, wherein: Reducing the proliferation and / or migration of vascular smooth muscle cells caused by the mechanical damage, thereby inhibiting the intimal neoplasia means that indole-3-propionic acid reduces the proportion of S phase cells in vascular smooth muscle cells and increases G0 / G1 and G2 / M phase arrest, thereby reducing the proliferation and migration of vascular smooth muscle cells, thereby inhibiting the intimal neoplasia of vascular smooth muscle cells.

6. A drug for treating and / or preventing restenosis after vascular intervention surgery, wherein: The drug includes indole-3-propionic acid.

7. The drug according to claim 6, wherein The medicament also includes pharmaceutically acceptable materials.

8. The drug according to claim 6 or 7, wherein The drug includes any one of an oral preparation, an injection and a surface coating agent, wherein the surface coating agent is coated on the surface of the stent and / or the balloon.

9. Use of indole-3-propionic acid in the preparation of a drug for reducing the expression of CDK2 in vascular smooth muscle cells.

10. Use of indole-3-propionic acid in screening drugs for treating and / or preventing restenosis after vascular interventional surgery; Preferably, the application includes a method for screening drugs, the screening method comprising: Treating vascular restenosis model animals or cells with the drug to be screened; Detect the proliferation of vascular smooth muscle cells in the treated vascular restenosis model animals or cells; Drug screening is performed based on the proliferation of the obtained vascular smooth muscle cells. If the drug to be screened reduces the proliferation of the vascular smooth muscle cells, the drug to be screened is the target drug obtained by screening.

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