Application of polypeptide MVAVA in preparation of medicine for preventing and treating endothelial cell injury

Abstract

The invention belongs to the technical field of endothelial cell injury treatment, and discloses application of polypeptide MVAVA in preparation of drugs for preventing and treating endothelial cell injury. In-vitro experiment results show that endothelial cell injury can be effectively inhibited when MVAVA treatment is carried out. The results show that the MVAVA polypeptide can be used as a novel polypeptide for resisting endothelial cell injury.

Description

Technical Field

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

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

[0003] Endothelial dysfunction is manifested by impaired endothelium-dependent vasodilation, increased oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Endothelial cell damage is considered to be a sign of the early development of a variety of cardiovascular and metabolic diseases, including atherosclerosis, hypertension, and diabetes. Therefore, targeted regulation of endothelial cell damage is an important strategy for the prevention and treatment of cardiovascular diseases and other diseases. Summary of the invention

[0004] The purpose of the present invention is to provide the use of polypeptide MVAVA in the preparation of drugs for preventing and / or treating endothelial cell damage. Studies have shown that polypeptide MVAVA or polypeptide MVAVA-TAT can be used to antagonize endothelial cell damage.

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

[0006] In a first aspect, the present invention seeks to protect the use of a polypeptide in the preparation of a drug for preventing and / or treating endothelial cell damage, wherein 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 (polypeptide MVAVA-TAT fused with the polypeptide MVAVA and the TAT membrane-penetrating peptide).

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

[0010] In a second aspect, the present invention seeks to protect a drug for preventing and / or treating endothelial cell damage, the active ingredient of which includes the above-mentioned polypeptide MVAVA or polypeptide MVAVA-TAT.

[0011] Beneficial effects of the present invention:

[0012] In vitro experiments showed that the peptide MVAVA-TAT could enter human umbilical vein endothelial cells after pretreatment, and could inhibit the increase of endothelial adhesion factor ICAM-1 / VCAM-1 expression induced by oxidized low-density lipoprotein (ox-LDL), and inhibit the ox-LDL-induced cell-cell junction disruption and monocyte adhesion. These results indicate that the MVAVA peptide can be used as a new anti-endothelial cell damage peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 By collecting plasma from subjects with coronary heart disease and healthy subjects for peptide omic testing, the differentially expressed peptide MVAVA was screened out.

[0014] Figure 2 The peptide MVAVA-TAT (50 μM) was added to human umbilical vein endothelial cells for pretreatment for 3, 6, 12, 24, and 48 hours, and then immunofluorescence staining and confocal microscopy were used to detect the membrane penetration efficiency of the peptide MVAVA-TAT on endothelial cells.

[0015] Figure 3 Human umbilical vein endothelial cells were pretreated with different concentrations of MVAVA-TAT (1, 10, 50, 100 μM) for 12 hours and then treated with ox-LDL (100 μM) for 24 hours. The expression of endothelial cell adhesion molecule ICAM1 / VCAM1 was detected by Western bolt; *<0.05.

[0016] Figure 4 After adding the peptide MVAVA-TAT (50 μM) to human umbilical vein endothelial cells, ox-LDL (100 μM) was given for 24 hours, and then the connections between endothelial cells were detected by VE-cadherin staining and confocal microscopy.

[0017] Figure 5 After adding the peptide MVAVA-TAT (50 μM) to human umbilical vein endothelial cells, ox-LDL (100 μM) was given for 24 hours and then the adhesion of monocytes to endothelial cells was detected by immunofluorescence staining and confocal microscopy. DETAILED DESCRIPTION

[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 are 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 purchased from GenScript Biopharmaceuticals.

[0019] SEQ ID NO.1: MVAVA

[0020] SEQ ID NO. 2: MVAVAYGRKKRRQRRR.

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

[0022] The plasma of subjects with coronary heart disease and healthy subjects was collected for peptide omic detection to screen differential peptides. Figure 1 Schematic diagram for screening out differentially expressed peptides MVAVA. In order to detect the transmembrane efficiency of MVAVA entering endothelial cells, we connected FITC (fluorescein isothiocyanate) to the N-terminus of the MVAVA peptide and the transmembrane peptide TAT (YGRKKRRQRRR) to the C-terminus, referred to as the peptide MVAVA-TAT. In order to study the transmembrane efficiency of the peptide MVAVA-TAT, we added the peptide MVAVA-TAT (50 μM) to human umbilical vein endothelial cells for pretreatment for 3, 6, 12, 24, and 48 hours, fixed them with paraformaldehyde for 20 minutes, added DAPI for staining for ten minutes, and used confocal microscopy to detect the transmembrane efficiency of the peptide MVAVA-TAT for endothelial cells. Figure 2 This is the membrane penetration efficiency diagram of the peptide MVAVA-TAT. It was found that the peptide could enter the endothelial cells in large quantities after pretreatment for 12 hours.

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

[0024] In order to detect the protective effect of peptide MVAVA on endothelial cell injury, we added different concentrations (1, 10, 50, 100 μM) of peptide MVAVA-TAT to human umbilical vein endothelial cells for pretreatment for 12 hours, and then treated them with ox-LDL (100 μM) for 24 hours to detect the expression of endothelial cell adhesion molecules ICAM1 / VCAM1. Figure 3Western bolt detection revealed that 50μM peptide MVAVA-TAT pretreatment for 12h could effectively inhibit the increase of adhesion factor ICAM1 / VCAM1 caused by ox-LDL. We then added peptide MVAVA-TAT (50μM) to human umbilical vein endothelial cells for pretreatment for 12h and then treated them with ox-LDL (100μM) for 24h. The cells were fixed with precooled methanol solution at room temperature (25±5℃) for 10min, blocked with 30mg / ml BSA solution for 60min, added VE-cadherin antibody and incubated overnight at 4℃, incubated with secondary antibody (anti-Mouse / Alexa Fluor 488) at room temperature for 60min, counterstained with DAPI solution (100nM) for nuclei, and observed intercellular junctions with laser confocal microscopy to evaluate endothelial integrity. Figure 4 The VE-cadherin fluorescence staining image shows that MVAVA-TAT pretreatment for 12 hours can effectively inhibit the ox-LDL-induced endothelial cell junction breakage. Human mononuclear THP-1 cells were stained with a cell membrane red fluorescence staining kit, and after 15 minutes of reaction, they were added to vascular endothelial cells and placed in a 37°C cell culture incubator for 60 minutes. After washing with 37°C phosphate buffer, the adhesion of THP-1 to endothelial cells was observed under a fluorescent inverted microscope and photographed. Figure 5 Fluorescence staining images showed that the peptide MVAVA-TAT could effectively inhibit the increase of monocyte adhesion caused by ox-LDL after 12h pretreatment.

Claims

1. Use of a polypeptide in the preparation of a drug for preventing and / or treating endothelial cell damage, characterized in that: The polypeptide has an amino acid sequence as 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 and / or treating endothelial cell damage, characterized in that: The active ingredient of the medicine comprises the polypeptide described in claim 1.

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