Application of polypeptide MVAVA in preparing drugs for preventing and treating endothelial cell injury

Abstract

The present invention belongs to the technical field of the treatment of endothelial cell injury, and discloses the application of polypeptide MVAVA in the preparation of a drug for preventing and treating endothelial cell injury. The results of in vitro experiments show that: by treating with MVAVA, the injury of endothelial cells can be effectively inhibited. These results indicate that the MVAVA polypeptide can be used as a new polypeptide for anti-endothelial cell injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of the treatment of endothelial cell injury, and particularly relates to the application of the polypeptide MVAVA for improving endothelial injury in the preparation of drugs for preventing and / or treating endothelial cell injury. Background Art

[0002] Vascular endothelium is composed of a continuous monolayer of cells covering the inner surface of the blood vessel lumen, and plays a crucial role in maintaining the health of multiple organs and homeostasis. Healthy endothelium can dynamically maintain vascular tone, angiogenesis, hemostasis, and provide an antioxidant, anti-inflammatory, and antithrombotic interface. The morphological and functional integrity of vascular endothelial cells (ECs) is necessary for normal blood vessels to maintain the normal functions of various tissues and organs of the body, and the integrity of endothelial function can antagonize the occurrence of various cardiovascular diseases.

[0003] Vascular endothelial dysfunction is manifested as impaired endothelium-dependent vasodilation function, elevated oxidative stress, chronic inflammation, leukocyte adhesion, high permeability, and endothelial cell senescence, etc. Endothelial cell injury is considered to be a sign of the early development of various cardiovascular and metabolic diseases, including atherosclerosis, hypertension, and diabetes, etc. Therefore, targeted regulation of endothelial cell injury is an important strategy for the prevention and treatment of diseases such as cardiovascular diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of the polypeptide MVAVA in the preparation of drugs for preventing and / or treating endothelial cell injury. Research shows that the polypeptide MVAVA or the polypeptide MVAVA-TAT can be used to antagonize endothelial cell injury.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] In the first aspect, the present invention claims the application of a polypeptide in the preparation of drugs for preventing and / or treating endothelial cell injury, and the polypeptide has an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] SEQ ID NO.1: MVAVA;

[0008] SEQ ID NO.2: MVAVAYGRKKRRQRRR (the polypeptide MVAVA-TAT, which is a fusion of the polypeptide MVAVA and the TAT cell-penetrating peptide).

[0009] Furthermore, in the above application, the active ingredient of the drug includes the aforementioned polypeptide MVAVA or the polypeptide MVAVA-TAT.

[0010] In a second aspect, the present invention claims protection for a drug for preventing and / or treating endothelial cell injury, and the active ingredient of the drug comprises the above-mentioned polypeptide MVAVA or polypeptide MVAVA-TAT.

[0011] Advantages of the present invention:

[0012] It can be seen from in vitro experiments that after pretreatment with polypeptide MVAVA-TAT, it can enter human umbilical vein endothelial cells, and can inhibit the increase in the expression of endothelial adhesion factors ICAM-1 / VCAM-1 induced by oxidized low-density lipoprotein (ox-LDL), and inhibit the disruption of cell-cell junctions and monocyte adhesion induced by ox-LDL. These results indicate that the MVAVA polypeptide can be used as a new polypeptide against endothelial cell injury. Brief Description of the Drawings

[0013] Figure 1 By collecting the plasma of coronary heart disease patients and healthy subjects for polypeptideomics detection, the differential polypeptide MVAVA was screened out.

[0014] Figure 2 After adding polypeptide MVAVA-TAT (50 μM) to human umbilical vein endothelial cells and pretreating for 3, 6, 12, 24, 48 h, the transmembrane efficiency of polypeptide MVAVA-TAT for endothelial cells was detected by immunofluorescence staining and confocal microscopy.

[0015] Figure 3 After adding different concentrations (1, 10, 50, 100 μM) of polypeptide MVAVA-TAT to human umbilical vein endothelial cells and pretreating for 12 h, ox-LDL (100 μM) was added and treated for 24 h, and the expression of endothelial cell adhesion molecules ICAM1 / VCAM1 was detected by Western blot; * < 0.05.

[0016] Figure 4 After adding polypeptide MVAVA-TAT (50 μM) to human umbilical vein endothelial cells and then treating with ox-LDL (100 μM) for 24 h, the endothelial cell-cell junctions were detected by VE-cadherin staining and confocal microscopy.

[0017] Figure 5 After adding polypeptide MVAVA-TAT (50 μM) to human umbilical vein endothelial cells and then treating with ox-LDL (100 μM) for 24 h, the adhesion of monocytes to endothelial cells was detected by immunofluorescence staining and confocal microscopy. Detailed Embodiments

[0018] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. The polypeptide MVAVA-TAT used in the examples is a synthetic product and was purchased from GenScript Biotech Corporation.

[0019] SEQ ID NO.1: MVAVA

[0020] SEQ ID NO.2: MVAVAYGRKKRRQRRR.

[0021] Example 1: Screening process and transmembrane efficiency of polypeptide MVAVA

[0022] Differential polypeptides were screened by collecting plasma from coronary heart disease patients and healthy subjects for polypeptide profiling. Figure 1 It is a schematic diagram for screening the differential polypeptide MVAVA. To detect the transmembrane efficiency of MVAVA entering endothelial cells, we linked the N-terminus of the MVAVA polypeptide to FITC (fluorescein isothiocyanate) and the C-terminus to the transmembrane peptide TAT (YGRKKRRQRRR), abbreviated as polypeptide MVAVA-TAT. To study the transmembrane efficiency of polypeptide MVAVA-TAT, we added polypeptide MVAVA-TAT (50 μM) to human umbilical vein endothelial cells and pretreated them for 3, 6, 12, 24, 48 h, then fixed them with paraformaldehyde for 20 minutes, dropped DAPI staining for ten minutes, and detected the transmembrane efficiency of polypeptide MVAVA-TAT for endothelial cells by confocal microscopy. Figure 2 It is the transmembrane efficiency diagram of polypeptide MVAVA-TAT. It was found that a large amount of the polypeptide could enter endothelial cells after 12 h of pretreatment.

[0023] Example 2: Protective effect of polypeptide MVAVA on endothelial cell injury

[0024] To detect the protective effect of polypeptide MVAVA on endothelial cell injury, we added different concentrations (1, 10, 50, 100 μM) of polypeptide MVAVA-TAT to human umbilical vein endothelial cells, pretreated them for 12 h, and then treated them with ox-LDL (100 μM) for 24 h to detect the expression of endothelial cell adhesion molecules ICAM1 / VCAM1. Figure 3Western blot detection revealed that pretreatment with 50 μM polypeptide MVAVA-TAT for 12 h could effectively inhibit the increase in adhesion factors ICAM1 / VCAM1 induced by ox-LDL. Subsequently, human umbilical vein endothelial cells were pretreated with polypeptide MVAVA-TAT (50 μM) for 12 h and then treated with ox-LDL (100 μM) for 24 h. The cells were fixed with pre-cooled methanol solution at room temperature (25 ± 5 °C) for 10 min, blocked with 30 mg / ml BSA solution for 60 min, incubated with VE-cadherin antibody at 4 °C overnight, incubated with secondary antibody (anti-Mouse / Alexa Fluor 488) at room temperature for 60 min, counterstained with DAPI solution (100 nM) for the nucleus, and the endothelial integrity was evaluated by observing cell junctions under a laser confocal microscope. Figure 4 The fluorescence staining map of VE-cadherin showed that pretreatment with MVAVA-TAT for 12 h could effectively inhibit the disruption of cell junctions between endothelial cells induced by ox-LDL. Human monocytes THP-1 were stained with a cell membrane red fluorescence staining kit. After reacting for 15 min, they were added to vascular endothelial cells and co-incubated in a 37 °C cell culture incubator for 60 min. After washing with phosphate buffer at 37 °C, the adhesion of THP-1 to endothelial cells was observed and photographed under a fluorescence inverted microscope. Figure 5 The fluorescence staining map showed that pretreatment with polypeptide MVAVA-TAT for 12 h could effectively inhibit the increase in monocyte adhesion induced by ox-LDL.

Claims

1. Use of a polypeptide in the preparation of a drug for preventing vascular endothelial dysfunction, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The application according to claim 1, characterized in that: The active ingredient of the medicine comprises the polypeptide described in claim 1.

3. A drug for preventing endothelial cell damage, characterized in that: The active ingredient of the medicine comprises the polypeptide described in claim 1.

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