Application of nuclear receptor binding protein 2 in treatment of postoperative restenosis neointimal hyperplasia

Abstract

The invention discloses application of nuclear receptor binding protein 2 in treatment of postoperative restenosis neointimal hyperplasia. The overexpressed nuclear receptor binding protein 2 can inhibit phenotypic transformation of vascular smooth muscle cells caused by platelet-derived growth factors (PDGF-BB) and enhancement of proliferation and migration capabilities. In addition, mouse carotid artery neointimal hyperplasia caused by carotid artery guide wire injury can be effectively improved through specific overexpression of the nuclear receptor binding protein 2 through smooth muscle cells, and it is prompted that the nuclear receptor binding protein 2 has a treatment effect and can serve as a marker to be applied to screening of drugs for treating postoperative restenosis neointimal hyperplasia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to application of nuclear receptor binding protein 2 in treating postoperative restenosis and neointimal hyperplasia. Background Art

[0002] Tissue ischemia and hypoxia caused by atherosclerotic lesions are important contributing factors to most cardiovascular diseases. Although the effects of stenting / angioplasty and drug-eluting stents in the treatment of atherosclerotic diseases have recently improved, the occurrence of restenosis after surgery makes the treatment effect unsatisfactory and increases the risk of recurrent cardiovascular events.

[0003] One common solution to postoperative restenosis is interventional therapy, such as percutaneous coronary angioplasty and stent implantation. These methods can help dilate the stenotic area and improve blood flow. However, these solutions have obvious shortcomings: although interventional therapy can effectively solve the problem of restenosis, the implementation process is complicated and may cause complications. In addition, patients still face the risk of restenosis after surgery, and such surgeries can only solve short-term problems and fail to fundamentally improve the problem. Another way is drug therapy, but long-term use of antibiotics and hormone drugs may cause side effects, such as gastrointestinal discomfort and bleeding. These drugs usually cannot completely eliminate the root causes of restenosis and neointimal hyperplasia, but only alleviate the symptoms to a certain extent.

[0004] Postoperative neointimal hyperplasia (NIH) is currently considered a key pathophysiological factor in restenosis. Neointimal hyperplasia (also known as neointimal formation) is a complex biological process mediated by a series of vasoactive substances and growth factors, involving functional changes in multiple cells, including endothelial cells, smooth muscle cells, and macrophages. In response to vascular injury and other stimuli, vascular smooth muscle cells (VSMCs) undergo abnormal proliferation, migration, and secretion of extracellular matrix, thereby forming a new neointimal membrane. During neointimal hyperplasia, various cells at the site of injury produce active factors such as platelet-derived growth factors (PDGFs) and TGF-β. Consequently, quiescent or contractile VSMCs begin to overexpress cell cycle-related genes, including PCNA and Cyclin D1, promoting cell growth. Overexpression of matrix metalloproteinases (MMPs) promotes cell migration into the arterial intima, leading to vessel wall thickening, lumen narrowing, or even occlusion. Therefore, maintaining the normal contractile state of VSMCs is a crucial prerequisite for preventing and treating postoperative restenosis. Studying the molecular mechanisms of VSMCs phenotypic transformation, proliferation, and migration will help to discover new intervention targets for neointimal hyperplasia.

[0005] Nuclear receptor binding protein 2 (NRBP2) is a conserved protein of 55-60 kDa that shares 59% amino acid similarity with nuclear receptor binding protein 1 (NRBP1). It was first identified by the Mammalian Gene Collection (MGC) process. The NRBP family was originally discovered to be involved in trafficking between the endoplasmic reticulum and the Golgi apparatus. Knockout of NRBP2 in zebrafish embryos by splicing-blocking morpholinos has been reported to result in global delay and embryonic lethality. NRBP2 expression in the brain is associated with neuronal differentiation in both normal and malignant brain tissue. However, no studies have yet reported on the role of NRBP2 in smooth muscle cell phenotypic transformation or vascular neointimal hyperplasia. Summary of the Invention

[0006] The current greatest technical challenge for treating neointimal hyperplasia associated with postoperative restenosis is the lack of effective long-term monitoring and intervention. Failure to effectively assess and manage patients after surgery can lead to restenosis and neointimal hyperplasia, increasing the patient's health burden. The present invention aims to provide the use of nuclear receptor binding protein 2 in the treatment of postoperative restenosis and neointimal hyperplasia.

[0007] The technical solution adopted by the present invention to solve the technical problem is:

[0008] In a first aspect, the present invention provides a use of protected nuclear receptor binding protein 2 in screening or assisting in screening drugs for treating postoperative restenosis and neointimal hyperplasia.

[0009] In a second aspect, the present invention provides a use of protected nuclear receptor binding protein 2 in the preparation of a drug for treating or assisting in treating postoperative restenosis and neointimal hyperplasia.

[0010] In a third aspect, the present invention protects the use of a product that overexpresses nuclear receptor binding protein 2 in the preparation of a drug for treating or assisting in the treatment of postoperative restenosis and neointimal hyperplasia.

[0011] In a specific embodiment, the product overexpressing nuclear receptor binding protein 2 is a recombinant vector containing a nuclear receptor binding protein 2 coding sequence.

[0012] In a specific embodiment, the recombinant vector includes but is not limited to: a plasmid, a phage, a cosmid, a Ti plasmid or a viral vector.

[0013] In a more specific embodiment, the recombinant vector is a lentivirus.

[0014] In a more specific embodiment, the shuttle plasmid used in constructing the lentivirus containing the nuclear receptor binding protein 2 coding sequence is pCDH-CMV-EGFP or pLVX-FLEX-FLAG-EF1a-ZsGree.

[0015] In a specific embodiment, the medicament further contains a pharmaceutically acceptable excipient.

[0016] In a fourth aspect, the present invention protects a drug for treating or assisting in treating postoperative restenosis and neointimal hyperplasia, wherein the drug comprises a recombinant vector containing a nuclear receptor binding protein 2 coding sequence.

[0017] In a fifth aspect, the present invention protects a method for treating or assisting in treating postoperative restenosis and neointimal hyperplasia, the method comprising administering an effective amount of nuclear receptor binding protein 2 to a patient.

[0018] Beneficial effects

[0019] The use of the nuclear receptor binding protein 2 provided by the present invention in treating postoperative restenosis and neointimal hyperplasia has the following beneficial effects compared with the prior art:

[0020] (1) The present invention found that the expression of nuclear receptor binding protein 2 was significantly reduced when a neointimal hyperplasia model was constructed at the cellular and animal levels.

[0021] (2) Overexpression of nuclear receptor binding protein 2 can inhibit the phenotypic transformation of vascular smooth muscle cells and the enhancement of proliferation and migration ability caused by platelet-derived growth factor (PDGF-BB); smooth muscle cell-specific overexpression of nuclear receptor binding protein 2 can effectively improve the neointimal hyperplasia of the mouse carotid artery caused by carotid artery guidewire injury, suggesting that nuclear receptor binding protein 2 plays a protective role in postoperative restenosis.

[0022] (3) This invention has developed a new method for preventing and treating postoperative restenosis and provided a meaningful reference for the development of new drugs for postoperative restenosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the expression of nuclear receptor binding protein 2 in the normal control model and the neointimal hyperplasia model. 8-week-old C57BL / 6J male mice were injured by guidewire in the left carotid artery to establish the neointimal hyperplasia model. The carotid artery vascular tissues of the mice were collected on the 7th, 14th and 28th days. Figure 1 A: Western blot analysis of the expression of nuclear receptor binding protein 2. *p<0.05. Figure 1 B: Western blot analysis of nuclear receptor binding protein 2 expression in human aortic smooth muscle cells (HASMC) treated with platelet-derived growth factor (25 ng / mL) for 36 h. *p < 0.05;

[0024] Figure 2 Schematic diagram of overexpression of nuclear receptor binding protein 2 to inhibit phenotypic transformation of smooth muscle cells. Lenti-control (Lenti-ctrl) and Lenti-NRBP2 (Lenti-NRBP2) lentivirus were added to human aortic smooth muscle cells (HASMC) for 36 hours, and then treated with platelet-derived growth factor (25 ng / mL) for 36 hours. Figure 2 A is western blot detection of the expression of smooth muscle cell contractile indicators a-SMA, CALPONIN, SM22, synthetic indicators VIMENTIN, OSTEOPONTIN and nuclear receptor binding protein 2. Figure 2 B is a quantitative graph, *p < 0.05;

[0025] Figure 3 Schematic diagram of overexpression of nuclear receptor binding protein 2 to inhibit smooth muscle cell proliferation and migration. Lentivirus encoding control (Lenti-ctrl) and nuclear receptor binding protein 2 (Lenti-NRBP2) was added to human aortic smooth muscle cells (HASMC) for 36 hours, and then platelet-derived growth factor (25 ng / mL) was treated. Figure 3 A: Scratch wound assay to detect the migration ability of smooth muscle cells. *p < 0.05. B: Transwell assay to detect the migration ability of smooth muscle cells. *p < 0.05.

[0026] Figure 4 Schematic diagram of overexpression of nuclear receptor binding protein 2 to improve postoperative restenosis and neointimal hyperplasia. Six-week-old male SM22 cre mice were injected with control (Lenti-ctrl) and nuclear receptor binding protein 2 (Lenti-NRBP2) lentivirus through the tail vein. Two weeks later, the left carotid artery was injured by guidewire. The left and right common carotid arteries were paraffin-embedded and sectioned for tissue staining. Figure 4 A is a schematic diagram of hematoxylin-eosin (HE) tissue staining to detect vascular neointimal hyperplasia. Figure 4 B is a quantitative graph, *P < 0.05;

[0027] Figure 5 Schematic diagram of overexpression of nuclear receptor binding protein 2 to inhibit smooth muscle cell phenotypic transformation in neointimal hyperplasia after restenosis. Vascular tissue proteins of the left and right common carotid arteries were extracted, and the expressions of smooth muscle cell contractile indicators a-SMA, CALPONIN, SM22, and nuclear receptor binding protein 2 were detected by western blot. *p<0.05. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the examples. Reagents or instruments used without manufacturer's indication are considered to be conventional products that can be purchased on the market.

[0029] The nuclear receptor binding protein 2 lentivirus used in the following examples was purchased from Wuhan Vinocell Biotechnology Co., Ltd.; wherein, the amino acid sequence of the nuclear receptor binding protein 2 protein is:

[0030] MAAPEPAPRRAREREREDESEDESDILEESPCGRWQKRREQVNQGNMPGLQSTFLAMDTEEGVEVVWNELHFGDRKAFAAHEEKIQTVFEQLVLVDHPNIVKLHKYWLDTSEACARVIFITEYV SSGSLKQFLKKTKKNHKAMNARAWKRWCTQILSALSFLHACSPPIIHGNLTSDTIFIQHNGLIKIGSVWHRIFSNALPDDLRSPIRAEREELRNLHFFPPEYGEVADGTAVDIFSFGMCALEMAVLE IQTNGDTRVTEEAIARARHSLSDPNMREFILCCLARDPARRPSAHSLLFHRVLFEVHSLKLLAAHCFIQHQYLMPENVVEEKTKAMDLHAVLAELPRPRRPPLQWRYSEVSFMELDKFLEDVRNGI YPLMNFAATRPLGLPRVLAPPPEEVQKAKTPTPEPFDSETRKVIQMQCNLERSEDKARWHLTLLLVLEDRLHRQLTYDLLPTDSAQDLASELVHYGFLHEDDRMKLAAFLESTFLKYRGTQA, as in SEQ ID NO:1.

[0031] The NCBI sequence number of the nucleotide sequence of the nuclear receptor binding protein 2 gene is 340371.

[0032] Example 1:

[0033] 1.1 Total protein extraction from cells / tissues

[0034] Cells: Wash cells twice with PBS, add cell lysis buffer prepared at a ratio of RIPA lysis buffer: protease inhibitor = 100:1, scrape the cells with a scraper and transfer them to an EP tube, and lyse on ice for 30 minutes; place tissue samples in EP tubes, add PBS, mince, and centrifuge at 4°C, 5000 rpm, and 10 minutes. Discard the supernatant, add cell lysis buffer, and transfer the tissue suspension to a tissue grinding tube. Grind in a freezer grinder for 60 seconds and let it stand on ice for 30 minutes; centrifuge at 4°C, 12000 rpm, and aspirate the supernatant. Detect protein concentration using a BCA kit. Add loading buffer to the protein supernatant, heat at 99°C in a metal bath for 5 minutes, and store in a -20°C refrigerator.

[0035] 1.2 Western Blot

[0036] After cleaning and mounting the glass plate, a 10% SDS-PAGE gel was prepared according to the instructions of the PAGE gel preparation kit. Samples were loaded, with 20 μg of protein added to each well. Electrophoresis was performed at a constant voltage of 75 V until the protein marker bands separated, then adjusted to a constant voltage of 115 V and electrophoresed to the bottom of the bromophenol blue gel. The PVDF membrane was activated with methanol for 1 min, and a sandwich structure was assembled in the order of sponge-filter paper-gel-PVDF membrane-filter paper-sponge. Black was placed on black, white on red, at a constant voltage of 100 V for 60 min. The membrane was then blocked with 10% skim milk solution at room temperature for 2 h. The PVDF membrane was cut according to protein molecular weight and incubated with primary antibodies overnight at 4°C (anti-NRBP2 rabbit polyclonal antibody, Proteintech, Catalog No. 21549-1-AP; anti-β-actin mouse monoclonal antibody, Abcam, Catalog No. ab6276). TBST was used. The membranes were washed three times for 5 minutes each time and incubated with secondary antibodies for 2 hours at room temperature on a shaker (HRP-labeled secondary antibody against rabbit, Jackson, catalog number: 111-035-003; HRP-labeled secondary antibody against mouse, Jackson, catalog number: 115-035-003). The membranes were washed three times with TBST for 5 minutes each time and developed using ECL colorimetric solution on an AI600 exposure imaging system. The grayscale values of the protein bands were calculated using ImageJ software.

[0037] 1.3 Establishment of an animal model of neointimal hyperplasia by guidewire injury of the common carotid artery

[0038] The mice were placed in a glass box connected to isoflurane gas for anesthesia, the head and limbs were fixed, the neck hair was removed, and the mice were disinfected with iodine. Use scissors to cut along the middle position of the mouse neck, separate the connective tissue near the common carotid artery on the left side of the mouse trachea and expose the common carotid artery, separate the connective tissue near the common carotid artery (CCA), internal carotid artery (IC), and external carotid artery (EC); tie a slipknot with 7-0 nylon thread at the common carotid artery and external carotid artery away from the bifurcation of the common carotid artery, tie a knot in the internal carotid artery, use microscissors to cut a small incision toward the vertical blood vessel from the knot of the internal carotid artery to the bifurcation of the common carotid artery, insert a vascular guide wire with a diameter of 0.35mm into the incision, extend the guide wire along the proximal end of the common carotid artery to injure the common carotid artery until the knot of the common carotid artery is reached; leave the guide wire in place for 5 minutes, and finally pull it back and forth 3 times, tie a knot from the incision to the bifurcation of the common carotid artery, untie the slipknot of the common carotid artery and external carotid artery, restore blood flow, and use 5-0 The neck skin of mice was sutured with nylon sutures; all mice in the experiment underwent guidewire injury on the left common carotid artery to establish a vascular neointimal hyperplasia model (NIH), and the right common carotid artery served as its own experimental control (Control).

[0039] 1.4 Platelet-derived growth factor-BB construction of neointimal hyperplasia cell model

[0040] Human aortic smooth muscle cells were treated with platelet-derived growth factor-BB (25 ng / ml) for 36 h to construct a neointimal hyperplasia cell model, and the changes in the transformation indicators of the corresponding smooth muscle cells were detected.

[0041] Example 2:

[0042] 2.1 Construction and infection of NRBP2 lentivirus:

[0043] (1) Construct NRBP2 lentivirus and select the shuttle plasmid pCDH-CMV-EGFP. The DNA sequence of NRBP2 in the NCBI GenBank library was used as the standard sequence (NCBI sequence number: 340371). After design and verification, the lentivirus was synthesized and packaged. Subsequently, the NRBP2 lentivirus was used to infect human vascular smooth muscle cells. After 48-72 hours, the green fluorescence intensity was observed by fluorescence microscopy to determine the NRBP2 infection efficiency. The expression efficiency of NRBP2 was then detected by Western blot technology, and subsequent experiments were carried out.

[0044] (2) Construction of NRBP2 lentivirus. The shuttle plasmid pLVX-FLEX-FLAG-EF1a-ZsGreen was selected. The DNA sequence of NRBP2 in the NCBI GenBank library was used as the standard sequence (NCBI sequence number: 340371). After design verification, the lentivirus was synthesized and packaged. Subsequently, the NRBP2 lentivirus was injected into the tail vein of 6-week-old SM22 cre male mice. 14 days later, the left carotid artery guidewire injury model was established. The green fluorescence intensity was observed by fluorescence microscopy to determine the NRBP2 infection efficiency. The expression efficiency of NRBP2 was then detected by Western blot technology. Subsequent experiments were then carried out.

[0045] 2.2 Transwell experiment

[0046] According to the cell number 1×10 5 Vascular smooth muscle cells were seeded in 8 μm Transwell chambers, which were placed in 24-well plates. 200 μl of FBS-free culture medium was added to the chambers; 500 μl of FBS-free culture medium was added to the well plate below the chambers, and the cells were cultured for 24 h after administration. The culture medium in the chambers was discarded, and the cells were washed twice with PBS. 600 μl of paraformaldehyde was added to the lower chamber and fixed at 4°C for 30 min. 600 μl of PBS was added to the lower chamber to wash away excess paraformaldehyde. The vascular smooth muscle cells were then stained with 1% crystal violet and placed at room temperature for 30 min. 600 μl of PBS was added to the lower chamber to wash away excess crystal violet, and the cells were photographed and counted under a microscope.

[0047] 2.3 Scratch test

[0048] Seed vascular smooth muscle cells in a six-well plate. When the cell density reaches about 90%, use a blue pipette tip to draw a line in the middle of the six-well plate along a ruler. Gently apply 1 ml of pre-warmed PBS to the side wall of the culture plate, gently shake the culture plate to wash away the cells scratched by the pipette tip, and then add 1 ml of culture medium. Give the corresponding stimulation treatment. Select a fixed field of view under the bright field of a microscope to observe and photograph the scratches. After 1 day of stimulation, gently apply 1 ml of pre-warmed PBS to the side wall of the culture plate, gently shake the culture plate, then add 1 ml of culture medium and photograph under bright field.

[0049] Here are the results:

[0050] 1. Under the pathological conditions of postoperative restenosis and neointimal hyperplasia, the expression of nuclear receptor binding protein 2 is significantly reduced. We performed left carotid artery guidewire injury in C57BL / 6 mice to establish a postoperative restenosis and neointimal hyperplasia model. Carotid artery vascular tissue was collected from mice on days 7, 14, and 28. Western blot analysis revealed that the expression of nuclear receptor binding protein 2 was significantly reduced in carotid artery vascular tissue ( Figure 1 A). In addition, we treated human aortic smooth muscle cells with platelet-derived growth factor-BB (25 ng / ml) for 36 hours to construct a neointimal hyperplasia cell model. Western Blot analysis revealed a significant decrease in the expression of nuclear receptor binding protein 2 ( Figure 1 B) These results indicate that NRBP2 expression is decreased in postoperative restenosis and neointimal hyperplasia, suggesting its involvement in the pathological process. NRBP2 may be a marker for postoperative restenosis and neointimal hyperplasia, potentially useful for screening drugs to treat postoperative restenosis.

[0051] 2. Overexpression of lentivirus and receptor-binding protein 2 at the cellular level inhibits phenotypic transformation of smooth muscle cells, and overexpression of lentivirus and receptor-binding protein 2 at the animal level improves neointimal hyperplasia in postoperative restenosis. We infected human aortic smooth muscle cells with lentivirus containing control (Lenti-ctrl) and nuclear receptor-binding protein 2 (Lenti-NRBP2) for 36 hours, then treated them with platelet-derived growth factor-BB (25ng / ml) for 36 hours. Western blot was used to detect the expression of smooth muscle cell contractile indicators a-SMA, CALPONIN, SM22, synthetic indicators VIMENTIN, OSTEOPONTIN, and lentivirus and receptor-binding protein 2. The results showed that overexpression of lentivirus and receptor-binding protein 2 could inhibit the decrease in smooth muscle cell contractile indicators and the increase in synthetic indicators caused by PDGF-BB. Figure 2 A. Figure 2 B). Scratch and transwell assays were used to detect the proliferation and migration of smooth muscle cells. The results showed that overexpression of nuclear receptor binding protein 2 could inhibit the enhancement of smooth muscle cell proliferation and migration induced by PDGF-BB ( Figure 3 Figure A, Figure 3 B) We selected 6-week-old SPF male SM22 cre mice and injected them with a control (Lenti-ctrl) or a Flag-tagged RNBP2 lentivirus into their tail veins. Two weeks later, carotid artery guidewire injury was performed for 4 weeks. Carotid artery vascular tissue was harvested for HE staining, and carotid artery tissue protein was collected. HE staining revealed that the area of neointimal hyperplasia in mice injected with the RNBP2 lentivirus into the tail vein was significantly smaller than that in mice injected with the control virus into the tail vein ( Figure 4 A, Figure 4B) By collecting the carotid artery vascular tissue proteins of mice, the results of western blot showed that the expression of contractile indicators of smooth muscle cells in mice injected with nuclear receptor binding protein 2 lentivirus in the tail vein was significantly higher than that in mice injected with the control virus in the tail vein ( Figure 5 A) These results suggest that NRBP2 plays a protective role in neointimal hyperplasia after restenosis, and overexpression of NRBP2 can delay the progression of the disease.

[0052] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. Application of nuclear receptor binding protein 2 in screening or assisting screening of drugs for the treatment of postoperative restenosis and neointimal hyperplasia.

2. Application of nuclear receptor binding protein 2 in the preparation of drugs for the treatment or auxiliary treatment of postoperative restenosis and neointimal hyperplasia.

3. Use of products that overexpress nuclear receptor binding protein 2 in the preparation of drugs for the treatment or adjuvant treatment of postoperative restenosis and neointimal hyperplasia.

4. The use according to claim 3, characterized in that The product for overexpressing nuclear receptor binding protein 2 is a recombinant vector containing a nuclear receptor binding protein 2 coding sequence.

5. The use according to claim 4, characterized in that The recombinant vector is a lentivirus.

6. The use according to claim 4, characterized in that The shuttle plasmid used to construct the lentivirus containing the nuclear receptor binding protein 2 coding sequence is pCDH-CMV-EGFP or pLVX-FLEX-FLAG-EF1a-ZsGree.

7. The use according to claim 3, characterized in that The medicine further contains pharmaceutically acceptable excipients.

8. A drug for treating or assisting in treating postoperative restenosis and neointimal hyperplasia, characterized in that: The medicine comprises a recombinant vector containing a nuclear receptor binding protein 2 coding sequence.

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