CN111671905B

CN111671905B - Application of P-selectin glycoprotein ligand-1 as target in preparation of drugs for preventing and/or treating aneurysms

Info

Publication number: CN111671905B
Application number: CN202010581519.9A
Authority: CN
Inventors: 杨志伟; 吴献贤; 刘星
Original assignee: Institute of Laboratory Animal Science of CAMS
Current assignee: Institute of Laboratory Animal Science of CAMS
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2022-05-27
Anticipated expiration: 2040-06-23
Also published as: CN111671905A

Abstract

The invention relates to the field of biological medicine, in particular to application of P-selectin glycoprotein ligand-1 as a target spot in preparation of a medicine for preventing and/or treating aneurysm. Compared with wild mice, PSGL-1 gene knockout mice are used as research objects, and the research results show that the incidence rates of thoracic aortic aneurysm and abdominal aortic aneurysm of the PSGL-1 gene knockout mice are obviously reduced, the collagen deposition is reduced, the phenomena of elastic fiber breakage and smooth muscle cell degradation are obviously inhibited, meanwhile, the infiltration of inflammatory cells is obviously reduced, and the expression of inflammatory factors, fibrosis factors and adhesion molecules is obviously inhibited. Furthermore, the adhesion of PSGL-1-depleted peripheral blood mononuclear cells to endothelial cells was also significantly reduced. The invention discloses that PSGL-1 deletion has the effect of inhibiting aneurysm, and provides theoretical basis and clinical basis for the function of PSGL-1 in research of new targets and new strategies for preventing and treating aneurysm.

Description

Application of P-selectin glycoprotein ligand-1 as target in preparation of drugs for preventing and/or treating aneurysms

Technical Field

The invention relates to the field of biological medicine, in particular to application of P-selectin glycoprotein ligand-1 as a target spot in preparation of a medicine for preventing and/or treating aneurysm.

Background

Aortic aneurysms can be classified into Thoracic Aortic Aneurysms (TAAs) and Abdominal Aortic Aneurysms (AAA) depending on the site of disease. TAA occurs in people of all ages, with similar incidence in both men and women, and is highly correlated with genetic factors. In contrast, AAA is usually associated with aging, male sex, atherosclerosis and smoking, but is less genetically related. AAA is the most common form of aortic aneurysm, common in adults over 50 years of age, and has a higher incidence in men than women. Aneurysm rupture is the most dangerous complication of abdominal aortic aneurysms and is the leading cause of death in patients. Currently, open surgical repair and endovascular repair are the only widely used therapies for treating aortic aneurysms, and no effective drugs for treating this destructive disease have been approved. Despite decades of research on AAA, there is a lack of understanding of the mechanisms and factors controlling AAA, and thus, elucidating the molecular basis of this disease, finding new therapeutic targets, and developing effective drugs that effectively inhibit the development of aneurysms remain important issues for aneurysm research.

Pathologically, AAA is characterized by expansion of the layers of the arterial wall, which can be attributed to loss of elastin, smooth muscle cell apoptosis, compensatory deposition of collagen, infiltration of inflammatory cells, oxidative stress, and the like. Previous studies have shown that inflammation is important in the development of aneurysms, the release of a series of proteolytic enzymes (e.g., matrix metalloproteinases and cysteine proteases) and the production of oxidative free radicals, inflammatory factors, caused by chronic aortic inflammation, are responsible for the destruction of the intima-media lining of the aorta and for the apoptosis and dysfunction of vascular smooth muscle cells. Inflammatory responses occur without infiltration of inflammatory cells, including T cells, B cells, macrophages, granulocytes. Macrophages are thought to be the major source of Matrix Metalloproteinases (MMPs) and proinflammatory cytokines. Experimental strategies to reduce the number of macrophages in aneurysms, such as by macrophage depletion or cytokine inhibition, can result in decreased MMP activity in AAA mouse models, reducing aneurysm formation. However, this method is cumbersome to implement and has poor clinical feasibility.

There is currently no effective pharmacological approach for the treatment of aneurysms, and open surgical repair and endovascular repair remain the only widely used therapies for the treatment of aortic aneurysms.

1. Use of open surgical repair and endovascular repair in the treatment of abdominal aortic aneurysms

Surgical treatment is the main strategy of current aneurysm treatment, the surgical aim being to excise the aneurysm or to reconstruct the artery. Since the first report by Parodi on endovascular aortic aneurysm repair (EVAR), the surgical approach to treating aneurysm disease has changed fundamentally. Although serious complications have occurred in the early application of EVAR, it still shows promise for surgical repair (OSR). EVAR has been widely used since the last two decades. In the united states, three-quarters of the currently available Abdominal Aortic Aneurysm (AAA) repairs are performed by endovascular methods. Despite the increasing popularity of EVAR, the choice between surgery and endovascular repair is still determined by the aneurysm and its potential for repair, the anatomical location, the size of the aneurysm and patient preference.

Open surgical repair and endovascular repair treatment of aneurysms: surgical treatment is indicated, and not all patients are suitable for open surgery or endovascular repair; surgical treatment is risky, with aneurysms themselves being a high risk disease, and the risks associated with surgical treatment are sometimes even higher than those associated with complications of aneurysms.

2. Study of drug therapy in prevention and treatment of abdominal aortic aneurysm

Currently, drugs for the treatment of abdominal aortic aneurysm, which are being clinically tested, include anti-inflammatory drugs, renin angiotensin system inhibitory drugs, antiplatelet drugs, and protease inhibitors.

1) Anti-inflammatory agents

Given the predominance of inflammation in AAA, several studies have attempted to mitigate aneurysm formation by targeting inflammatory cell types including macrophages and T lymphocytes. Inhibition of aneurysm formation by targeting inflammatory factors, including IL, TNF- α and TGF- β, has also been investigated. However, many of the therapies used in these animals are not approved for use in humans. To accelerate the transformation of preclinical findings, several studies examined the effect of clinically recognized immunosuppressive drugs including cyclosporine, infliximab, and azathioprine on AAA formation. Although these studies have shown promising results in attenuating aneurysm formation, the clinical application of these anti-inflammatory agents should be prudent because of their broader immunosuppressive capabilities.

2) Renin angiotensin system inhibitory drug

Many retrospective clinical studies have investigated the efficacy of ACE inhibitors or ARBs in limiting AAA progression. In these studies, there is debate regarding the efficacy of ACE inhibitors. Hackam et al demonstrate a reduced likelihood of breakage in patients prescribed ACE inhibitors. While the AAA-related scholars in the uk reported an increase in AAA growth rate in ACE-inhibited patients. Retrospective ARB studies show that ARB is associated with a decrease in AAA progression. Currently, several large random control trials are underway, aiming to study the effect of ACE inhibition [ AARDVARK (NCT01118520) ] or ARB [ TEDY (NCT01683084) and (NCT01904981) ] on AAA growth rate.

3) Antiplatelet drugs

Approximately 70% to 80% of patients with AAA present non-obstructive endoluminal thrombi (ILT) located in the vessel wall, which ILT communicates with the circulating blood. ILT is a determinant of AAA integrity and aneurysm growth and may be biologically important. Early thrombus formation is a complex metabolically active structure that contains activated platelets, a small number of white blood cells (primarily neutrophils, called polymorphonuclear cells), a thick fibrin network and encapsulated red blood cells.

Most thrombi contain a three-layered morphology, which originates from persistent transient neutrophil apoptosis, erythrolysis and the deposition of platelets and fibrin. It follows that antithrombotic therapy has become a potential drug for the treatment of AAA progression. However, the efficacy of these treatments has not been demonstrated, and the effect of reducing ILT may be disadvantageous.

4) Protease inhibitors

Matrix Metalloproteinases (MMPs) are involved in the pathogenesis of aortic aneurysms, as the histologic features of Thoracic Aortic Aneurysms (TAAs) and Abdominal Aortic Aneurysms (AAA) are loss of smooth muscle cells in the aortic media, degradation of elastic fibers, and disruption of the extracellular matrix (ECM). These pathological changes collectively result in a localized dilatation of the aorta. MMPs, which have many subtypes, among which MMP9 synthesized and secreted by macrophages and MMP2 secreted by smooth muscle cells play an important role in the development of aneurysm occurrence, degrade extracellular matrix. Inhibitors targeting MMPs show a positive effect in delaying the development of aneurysms.

The existing potential drugs for treating aneurysm have the following disadvantages:

the existing medicines are concentrated at the animal test or clinical test stage at present, and some medicines have controversial results in the clinical test stage, the target inflammatory cell and inflammatory factor therapy aims at the terminal link of inflammatory reaction, the inflammation is not controlled from the initial stage of inflammatory reaction, only the effect of delaying and weakening can be achieved, and the generation of aneurysm cannot be fundamentally inhibited. Protease inhibitor drugs also act against established proteases and do not inhibit the initial steps of aneurysm development, while antithrombotic drugs are only effective in inhibiting aneurysms containing thrombi, and the therapeutic effects of antithrombotic therapies require clinical trial and development. Although the renin angiotensin system inhibitory drugs have been clinically tested, the results of the clinical tests are not completely consistent, and the therapeutic effects on the aneurysm are controversial.

In conclusion, despite the research on AAA for decades, the pathogenesis and factors of AAA are poorly understood, and thus, it is of great practical significance to the research on aneurysm to elucidate the molecular basis of the disease, find new therapeutic targets, and develop effective drugs that effectively inhibit the development of aneurysm.

Disclosure of Invention

In view of the above, the present invention provides an application of P-selectin glycoprotein ligand-1 as a target in the preparation of a medicament for preventing and/or treating aneurysm.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of P-selectin glycoprotein ligand-1 as a target in preparation of a medicament for preventing and/or treating aneurysm.

In some embodiments of the invention, the aneurysm is an aortic aneurysm; the aortic aneurysm includes a thoracic aortic aneurysm and/or an abdominal aortic aneurysm.

In some embodiments of the invention, deletion, inhibition, or inactivation of P-selectin glycoprotein ligand-1 inhibits the development or progression of an aneurysm.

In some embodiments of the invention, deletion, inhibition, or inactivation of P-selectin glycoprotein ligand-1 inhibits inflammatory cell infiltration and/or expression of proinflammatory factors; deletion, inhibition, or inactivation of P-selectin glycoprotein ligand-1 promotes the expression of anti-inflammatory factors;

the inflammatory cells comprise one or more of CD3+ T cells, CD68+ macrophages or ly6b.2 neutrophils;

the proinflammatory factors comprise one or more of TNF-alpha, IL-6, IL-1 beta or CCL-2;

the anti-inflammatory factor comprises IL-10 and/or IL-13.

In some embodiments of the invention, the deletion, inhibition, or inactivation of P-selectin glycoprotein ligand-1 inhibits the adhesion of peripheral blood mononuclear cells to endothelial cells.

On the basis of the research, the invention also provides application of the inhibitor of the P-selectin glycoprotein ligand-1 in preparing a medicament for preventing and/or treating aneurysm.

In some embodiments of the invention, the inhibitor comprises an RNA interference vector of immune-related PSGL-1, a monoclonal antibody to immune-related PSGL-1 and/or other inhibitors capable of inhibiting PSGL-1 expression or blocking binding of PSGL-1 to P/E selectin.

The invention also provides a medicament for preventing and/or treating aneurysm, which comprises an inhibitor of P-selectin glycoprotein ligand-1. In some embodiments of the invention, the inhibitor comprises an RNA interference vector of immune-related PSGL-1, a monoclonal antibody to immune-related PSGL-1 and/or other inhibitors capable of inhibiting PSGL-1 expression or blocking binding of PSGL-1 to P/E selectin.

In addition, the invention also provides application of the PSGL-1 gene as a target gene in preparation of biological reagents and/or medicines for preventing and/or treating aneurysm. The PSGL-1 gene can be used as a target point to construct an in vitro cell model or an animal model of PSGL-1 deletion, and the in vitro cell model or the animal model can be used for screening drugs for treating or preventing aneurysms. PSGL-1 gene can be used as target gene in gene therapy, and can be used for designing and preparing related biological reagent, and can be used for preventing or curing diseases by means of gene engineering technology.

Leukocyte binding and rolling are the initial steps in the development of an inflammatory response, and in vivo binding and rolling of inflamed vessels is mediated primarily by selectins (selectins). PSGL-1 is a transmembrane glycoprotein constitutively expressed on the surface of leukocytes and is a selectin-binding ligand. PSGL-1 plays a key role in the adhesion and infiltration of inflammatory cells by mediating early adhesion and initial rolling of leukocytes to the endothelium. At present, no research reports the relation between PSGL-1 and aneurysm occurrence, and the invention firstly proves that the expression of PSGL-1 in aneurysm tissue is obviously increased, and PSGL-1 can mediate the adhesion and infiltration of inflammatory cells in the process of aneurysm occurrence. The invention provides and proves that PSGL-1 is taken as a target for regulating the generation and development of the aneurysm, fills the blank in the field that PSGL-1 influences the generation and the rupture of the aneurysm in domestic and foreign researches, and provides a new treatment target for developing a medicament for effectively inhibiting the generation and the development of the aneurysm.

Thus, the present invention solves the following problems: 1) PSGL-1 can be used as a therapeutic target of aneurysm, and the inhibition of PSGL-1 can control the generation and development of aneurysm; 2) the PSGL-1 inhibitor comprises RNA interference vectors of immune-related PSGL-1, monoclonal antibodies of the immune-related PSGL-1 and other inhibitors capable of inhibiting the expression of PSGL-1 or blocking the combination of PSGL-1 and P/E selectin, and can inhibit the generation and development of aneurysm; 3) PSGL-1 inhibitors protect against the development of aneurysms by inhibiting the adhesion of peripheral blood mononuclear cells to endothelial cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the structure of human PSGL-1 protein;

FIG. 2 shows that PSGL-1 expression is significantly elevated in human aneurysm tissue and mouse aneurysm tissue; wherein, FIG. 2A shows the protein expression of PSGL-1 in human aortic aneurysm samples; FIG. 2B shows protein expression of PSGL-1 in human aortic dissection samples; FIG. 2C shows an increase in protein expression level of PSGL-1 in human aortic aneurysm samples; FIG. 2D shows an increase in protein expression level of PSGL-1 in human aortic dissection samples; FIG. 2E shows increased levels of PSGL-1 protein expression in DOCA plus salt induced mouse aneurysm tissue; FIG. 2F shows increased expression levels of PSGL-1 mRNA in DOCA plus salt induced mouse aneurysm tissues; FIG. 2G shows angiotensin II (AngII) -induced increase in PSGL-1 mRNA expression levels in mouse aneurysm tissues;

FIG. 3 shows the protective effect of PSGL-1 deletion on aneurysms; wherein, fig. 3A shows the abdominal aorta ultrasound results; FIG. 3B shows the formation of hemangiomas in each group; FIG. 3C shows statistics of the outer diameters of various groups of abdominal aorta; FIG. 3D shows abdominal aortic aneurysm, thoracic aortic aneurysm tumorigenesis rate, and mortality rate for each group of mice; FIG. 3E shows the collagen deposition, elastin cleavage and smooth muscle cell degradation in arterial tissue of various groups of mice; FIG. 3F shows matrix metalloproteinase and collagen expression in aortic tissue of various groups of mice;

FIG. 4 shows that PSGL-1 deletion inhibits inflammatory cell infiltration and inflammatory factor expression; wherein, FIG. 4A shows the infiltration of CD3+ T cells, CD68+ macrophages and Ly6B.2 granulocytes in each group of arterial tissues; FIG. 4B shows the proinflammatory factor TNF- α mRNA expression levels in various groups of arterial tissue; FIG. 4C shows the proinflammatory factor IL-6 mRNA expression levels in various groups of arterial tissue; FIG. 4D shows proinflammatory factor IL-1 β mRNA expression levels in groups of arterial tissue; FIG. 4E shows the proinflammatory factor CCL-2 mRNA expression levels in various groups of arterial tissue; FIG. 4F shows anti-inflammatory factor IL-13 mRNA expression levels in various groups of arterial tissues; FIG. 4G shows anti-inflammatory factor IL-10 mRNA expression levels in various groups of arterial tissues;

FIG. 5 shows that PSGL-1 deletion inhibits adhesion of peripheral blood mononuclear cells to endothelial cells; wherein, FIG. 5A shows the expression of PSGL-1 on the surface of peripheral blood leukocytes of each group; FIG. 5B is a schematic diagram showing an in vitro static monocyte-endothelial cell adhesion experiment; FIG. 5C shows leukocyte and endothelial cell adhesion under different treatment conditions.

Detailed Description

The invention discloses application of P-selectin glycoprotein ligand-1 as a target in preparing a medicament for preventing and/or treating aneurysm, and the technical personnel can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate variations and combinations of the methods and applications described herein may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Interpretation of terms:

aortic aneurysm: aortic aneurysms can be classified into Thoracic Aortic Aneurysms (TAAs) and Abdominal Aortic Aneurysms (AAA) depending on the location of the disease. TAA occurs in people of all ages, has no gender association, and is highly correlated with genetic factors. In contrast, AAA occurs in association with age, gender, atherosclerosis, and smoking, but is less genetically related. AAA is the most common form of aortic aneurysm, common in adults over 50 years of age, and has a higher incidence in men than women. Aneurysm rupture is the most dangerous complication of abdominal aortic aneurysms and is the leading cause of death in patients.

Inflammatory reaction: inflammatory responses include infectious inflammatory responses, which are typically caused by microbial infection, and sterile inflammatory responses, which are caused by a wide variety of sterile particles, with significant fibrosis in some cases. These particles include particles whose composition is inorganic (e.g. silica, iron oxide, calcium pyrophosphate, asbestos) and organic (e.g. monosodium urate, amyloid β, cholesterol), whose structure may be crystalline (e.g. silicates, sodium urate) or amorphous (e.g. alum and iron oxide). The inflammatory reaction is a complex pathological process, and in the case of cardiovascular diseases, the inflammatory reaction is a nonspecific sterile immune reaction and comprises infiltration of inflammatory cells, secretion of inflammatory factors, damage of the inflammatory factors to target organs and the like.

P-selectin glycoprotein ligand-1 (PSGL-1): leukocyte binding and rolling are the initial steps in the development of an inflammatory response, and in vivo binding and rolling of inflamed vessels is mediated primarily by selectins (selectins). P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-type glycoprotein ligand that is expressed persistently on the surface of all leukocytes (including monocytes, granulocytes, lymphocytes and some CD34+ stem cells) and is capable of binding to selectin. There are three subtypes of selectins, including P-selectin, E-selectin, and L-selectin, where P-selectin is expressed on the surface of activated platelets and vascular endothelial cells, E-selectin is expressed on the surface of activated endothelial cells, and L-selectin is expressed on the surface of most leukocytes. PSGL-1 binds to three selectins, but binds most strongly to P-selectin. After PSGL-1 is combined with activated vascular endothelial surface P-selectin/E-selectin, adhesion and rolling of leucocytes and vascular endothelium are started, infiltration of leucocytes to intima-membrane is promoted, and secretion of inflammatory factors is promoted; PSGL-1 binds to P-selectin on the surface of activated platelets, leading to the formation of leukocyte-platelet complexes, further promoting the adhesion and infiltration of inflammatory cells; the combination of PSGL-1 and L-selectin on the surface of T cells can regulate the homing of T cells and promote the secretion of subsequent inflammatory factors.

The structure of human PSGL-1 protein includes a binding region, a transmembrane region and a cytoplasmic region (see FIG. 1, PSGL-1as an extracellular target. Constantin G. drug News Perspectrum 2004). Mucins form disulfide-bonded homodimers thereon. Human PSGL-1 is rich in serine, threonine and proline and contains 15 decamer repeats. The three NH2 terminal tyrosines of

residues

46, 48 and 51 are located in the anionic consensus sequence favouring tyrosine sulfation. Potential O-linked glycosylation sites are present in

threonine

44, 57, 69 and 70. P-selectin binds to the N-terminus of PSGL-1 by stereospecific interaction with clustered tyrosine sulfate and nearby core 2O-glycans, with sialylated Lewis x (sLex) epitopes. Similarly, L-selectin binds with high affinity to the N-terminal region of PSGL-1 through a synergistic interaction with three sulfated tyrosine residues and the appropriately positioned C2-O-sLex O-glycan. Binding of E-selectin-PSGL-1 appears to be independent of sulfation, requiring fucosyltransferases to glycosylate sLex and PSGL-1.

Compared with wild mice, PSGL-1 gene knockout mice are used as research objects, and the research results show that the incidence rates of thoracic aortic aneurysm and abdominal aortic aneurysm of the PSGL-1 gene knockout mice are obviously reduced, the collagen deposition is reduced, the phenomena of elastic fiber breakage and smooth muscle cell degradation are obviously inhibited, meanwhile, the infiltration of inflammatory cells is obviously reduced, and the expression of inflammatory factors, fibrosis factors and adhesion molecules is obviously inhibited. Furthermore, the adhesion of PSGL-1-depleted peripheral blood mononuclear cells to endothelial cells was also significantly reduced. The invention discloses that PSGL-1 deletion has the effect of inhibiting aneurysm, and provides theoretical basis and clinical basis for the function of PSGL-1 in research of new targets and new strategies for preventing and treating aneurysm.

Starting from the initial stage of inflammatory cell infiltration, the invention inhibits the infiltration of inflammatory cells by inhibiting PSGL-1, further inhibits the release of inflammatory factors and the release of metalloproteases secreted by the inflammatory cells, blocks important factors of aneurysm formation from the initial stage, and inhibits the occurrence and development of aneurysm, rather than simply weakening the progress of the aneurysm.

Therefore, the PSGL-1 gene is used as a target point to construct an in vitro cell model or an animal model with PSGL-1 deletion, and the in vitro cell model or the animal model is used for screening drugs for treating or preventing aneurysm; PSGL-1 gene can also be used as target gene in gene therapy, design and prepare related biological reagents, and achieve the purpose of prevention or treatment through gene engineering technology; the PSGL-1 protein can also be used as an immunological target to prepare a PSGL-1 monoclonal antibody, thereby achieving the purposes of blocking the function of PSGL-1, weakening the adhesion of inflammatory cells and treating aneurysm.

The P-selectin glycoprotein ligand-1 provided by the invention is used as a target point in the application of preparing a medicament for preventing and/or treating aneurysm, and the adopted raw materials and reagents can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 preparation of mouse animal model for aneurysm

The invention adopts 10-month-old PSGL-1^+/+Mouse and PSGL-1^-/-Mice were given deoxycorticosterone acetate (DOCA) plus saline water to induce the development of aneurysms. DOCA sustained release tablets: 50mg, released over 21 days. High salt content: 0.9% NaCl plus 0.2% KCl. After anesthetizing the mice, the mice were shaved on their backs and then all the hair at the surgical site was removed using a depilatory cream. The skin on the outside of the animal's neck was lifted, a cut was made to match the size of the tablet, a pocket was made horizontally with a pair of tweezers of about 2 cm just above the cut, and the DOCA tablet was placed into the pocket with the tweezers. The wound was closed with a wound clip and sutured. Mice were started to consume high-salt drinking water (0.9% NaCl plus 0.2% KCl) for 3 weeks immediately after DOCA implantation.

Example 2 ultrasonic detection of aortic internal diameter in Small animals

Using a high resolution ultrasound imaging system (Vevo 2100, Visualsonics, Toronto, Canada)The internal diameter of the abdominal aorta lumen above the mouse kidney was imaged and quantified. Mice were anesthetized by isoflurane cocktail inhalation, using VIP 3000 isoflurane matrix with O during the entire procedure₂(3-5% isoflurane/97% O2) and by inhalation isoflurane admixed with O2 (1-3% isoflurane/97% O)₂) And maintaining. Hair was removed from the mice from the abdomen using depilatory cream. Mice were supine on a heated table and a warm ultrasound transmitting gel was applied to the abdomen. Ultrasound images are taken over the entire region of the abdominal aorta and used to determine the maximum diameter of the abdominal aorta in the suprarenal region.

Example 3 pathological assay

1) HE staining: dewaxing the cut abdominal aorta paraffin section until water treatment, then staining with hematoxylin for 5min, and washing with tap water; ethanol hydrochloride is differentiated for 30 sec; flushing with running water, or returning blue with dilute ammonia water for 5 sec; eosin staining for 5min, washing with tap water; dehydrating, removing alcohol, and sealing.

2) Masson staining: dewaxing the cut abdominal aorta paraffin section until water treatment is carried out, reacting with Bouin liquid for one night at room temperature, and then washing with running water; staining with celestite blue staining solution for 2min, slightly washing with hematoxylin for 2min, slightly washing with water, and differentiating with acidic ethanol differentiation solution for several seconds; washing with running water for 10min, dyeing with plum-spring red magenta dyeing solution for 10min, and washing with distilled water; treating with phosphomolybdic acid solution for about 10min, pouring out supernatant, slicing without washing with water, and directly dripping aniline blue dyeing solution for dyeing for 5 min; treating with weak acid solution for 2 min; dehydrating, removing alcohol, and sealing.

3) Dyeing elastic fibers: paraffin sections were dewaxed to 95% ethanol, stained with weigert magenta stain for 2h, and washed slightly with tap water. Differentiating with 95% ethanol, and washing with water for 5 min. The prepared VG staining solution is used for counterstaining for 30 s. Differentiation was rapidly carried out with 95% ethanol. Dehydrated by absolute ethyl alcohol, transparent by xylene and sealed by neutral gum.

4) And (3) immunofluorescence staining: freezing and slicing arterial tissue, fixing with 4% paraformaldehyde at room temperature for 30min, and washing with PBS for three times; sealing goat serum for 1 h; adding primary antibody, and standing overnight at 4 deg.C; adding a secondary antibody, carrying out immersion washing with PBS at room temperature for 1 h; sealing the film by using DAPI staining solution, and imaging by using a laser confocal system.

Example 4 leukocyte adhesion assay

Peripheral blood mononuclear cells were isolated from peripheral blood of mice using Histopaque-1077 isolate, then labeled with 5 μ M calciin-AM at 37 ℃ for 30 minutes, and then washed 3 times with PBS. The labeled PBMC were then co-cultured with Bend.3 cells for 1h to allow cell adhesion. The PBMC/endothelial cell mixture was washed 3 times with PBS. The adhered PBMCs were photographed with a microscope and the relative fluorescence intensity was calculated with Image J software.

Example 5 RT-PCR

Total RNA was extracted from aortic tissue using Trizol reagent (Invitrogen, CA) and PrimeScriptTM RT MasterMix (RR036A, TaKaRa) was transcribed into cDNA. The cDNA was amplified with SYBR GreenPCRMastermix (applied biosystems, Calif.) and detected using 7500 Fast Real-time PCR Systemmachine (applied biosystems, Calif.).

Example 6 Western blot

Total protein was extracted from aortic tissues using RIPA buffer containing protease inhibitors and then its concentration was determined by BCA protein assay kit. Equal amounts of protein samples were loaded onto SDS-PAGE and then transferred onto nitrocellulose membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4 ℃ with primary antibodies against PSGL-1 and GAPDH, followed by secondary antibodies. Protein bands were imaged by the Tanon 5500 chemiluminescence imaging system.

Effect example 1 PSGL-1 expression was significantly elevated in human aneurysm tissue and mouse aneurysm tissue

As shown in fig. 2: the expression of PSGL-1 protein in a human aortic aneurysm tissue sample and a human aortic dissection tissue sample is obviously increased compared with that in a normal control group; the content of PSGL-1 protein and mRNA in the aortic aneurysm tissue of the mouse is obviously increased compared with that of the control mouse, and the PSGL-1 can be used as a marker for the occurrence of the aneurysm.

TABLE 1 FIG. 2C data

Table 2 figure 2D data

Table 3 fig. 2F data

Effect example 2 inhibition of aneurysm by PSGL-1 deletion

As shown in fig. 3: after PSGL-1 gene knockout, DOCA is also given for high-salt induction, but the inner diameter and the outer diameter of the aorta of the mouse are obviously lower than those of a wild type mouse, and the thoracic aortic aneurysm, the abdominal aortic aneurysm and the death rate are also obviously reduced; pathological staining results show that PSGL-1 deletion inhibits collagen deposition, elastic fiber breakage and smooth muscle cell degradation; further detecting the expression of fibrosis related factors in tissues to find that PSGL-1 lacks and inhibits the expression of the fibrosis factors; therefore, the PSGL-1 deletion inhibits the generation and the development of the aneurysm, and the inhibition of PSGL-1 expression has a protective effect on the aneurysm.

Table 4 fig. 3C data

Table 5 fig. 3C data analysis

Table 6 figure 3D data

Grouping	Number of	TAA	AAA	Death
					PSGL-1^+/+(HS)	15	0(0％)	0(0％)	0(0％)
PSGL-1^+/+(DOCA+HS)	23	11(48％)	15(65％)	8(35％)
					PSGL-1^-/-(HS)	10	0(0％)	0(0％)	0(0％)
PSGL-1^-/-(DOCA+HS)	15	2(3％)	2(13％)	0(0％)

With respect to TAA:

PSGL-1^+/+(HS)VS PSGL-1^+/+(DOCA + HS) chi fang 10.1, P0.0015, chi fang test

PSGL-1^+/+(DOCA+HS)VS PSGL-1^-/-(DOCA + HS) chi fang 4.799, P0.0285, chi fang test

With regard to AAA:

PSGL-1^+/+(HS)VS PSGL-1^+/+(DOCA + HS) that chi Fang ═ 16.16, P < 0.0001

PSGL-1^+/+(DOCA+HS)VS PSGL-1^-/-(DOCA + HS) chi Fang 9.886, p 0.0017

Regarding the death:

PSGL-1^+/+(HS)VS PSGL-1^+/+(DOCA + HS) that chi Fang (6.609), P (0.0101)

PSGL-1^+/+(DOCA+HS)VS PSGL-1^-/-(DOCA + HS): chi fang is 6.609, P is 0.0101.

Table 7 fig. 3F data

Effect example 3 deletion of PSGL-1 inhibits inflammatory cell infiltration and inflammatory factor expression

As shown in fig. 4: compared with wild mice, the infiltration inflammatory cells (CD3+ T cells, CD68+ macrophages, Ly6B.2 neutrophils) in artery tissues of the PSGL-1 knockout mice are obviously reduced, the expression of proinflammatory factors (TNF-alpha, IL-6, IL-1 beta, CCL-2) is obviously reduced, and the expression of anti-inflammatory factors (IL-10 and IL-13) is obviously increased, which shows that the protective effect of PSGL-1 deletion on aneurysm is closely related to inflammation inhibition.

TABLE 8 FIG. 4B data

TABLE 9 FIG. 4B data analysis

TABLE 10 FIG. 4C data

Table 11 fig. 4C data analysis

Table 12 fig. 4D data

Table 13 fig. 4D data analysis

Table 14 fig. 4E data

Table 15 fig. 4E data analysis

Table 16 fig. 4F data

TABLE 17 FIG. 4F data analysis

Table 18 fig. 4G data

TABLE 19 FIG. 4G data analysis

Effect example 4 deletion of PSGL-1 inhibits adhesion of peripheral blood mononuclear cells to endothelial cells

As shown in fig. 5: PSGL-1 on the surface of Peripheral Blood Mononuclear Cells (PBMCs) of a PSGL-1 knockout mouse is deleted, and cell adhesion experiments show that the adhesion capability of the PBMCs without the PSGL-1 and leukocytes is obviously reduced, and the number of adhered cells is obviously reduced, so that the protective effect of the PSGL-1 deletion on aneurysm is realized by inhibiting the adhesion of the leukocytes and endothelial cells, namely inhibiting the initial step of inflammatory reaction, and then inhibiting inflammatory cell infiltration and the release of inflammatory factors.

Table 20 fig. 4A data

Table 21 fig. 4A data analysis

Table 22 fig. 4C data

Table 23 fig. 4C data analysis

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

Use of deletion, inhibition or inactivation of P-selectin glycoprotein ligand-1 in the manufacture of a medicament for the prevention and/or treatment of an aneurysm.
2. The use of claim 1, wherein the aneurysm is an aortic aneurysm; the aortic aneurysm includes a thoracic aortic aneurysm and/or an abdominal aortic aneurysm.
3. The use of claim 1 or 2, wherein the deletion, inhibition or inactivation of P-selectin glycoprotein ligand-1 inhibits inflammatory cell infiltration and/or proinflammatory factor expression;

the inflammatory cells comprise one or more of CD3+ T cells, CD68+ macrophages or ly6b.2 neutrophils;

the proinflammatory factors include one or more of TNF-alpha, IL-6, IL-1 beta or CCL-2.
4. The use of claim 3, wherein the deletion, inhibition, or inactivation of P-selectin glycoprotein ligand-1 inhibits adhesion of peripheral blood mononuclear cells to endothelial cells.