Application of macrophage Angulin-1 in preparation of drugs and diagnostic products for preventing and / or treating atherosclerosis and related diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a macrophage Angulin-1 gene and / or an Angulin-1 protein in preparation of medicines and diagnostic products for preventing and / or treating atherosclerosis and related diseases. Research finds that plaque and necrotic core areas of an atherosclerosis animal model are remarkably increased due to macrophage Angulin-1 gene knockout, and disease development is promoted; and the recovery of the expression level of Angulin-1 significantly inhibits the development of lesion. Cell experiments show that Angulin-1 reduces intake of oxidized low-density lipoprotein by down-regulating expression of macrophage LOX-1, so that formation of foam cells is inhibited. Therefore, the macrophage Angulin-1 can be used as an atherosclerosis drug target, a gene therapy target gene and an auxiliary diagnosis marker, and a new theoretical basis and an intervention strategy are provided for prevention and treatment of the disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of macrophage Angulin-1 gene and / or Angulin-1 protein in the preparation of drugs and diagnostic products for preventing and / or treating atherosclerosis and related diseases. Background Art

[0002] Atherosclerosis is a major cause of high morbidity and mortality in cardiovascular and cerebrovascular diseases. The transformation of macrophages into foam cells is a key component of the pathological process of atherosclerosis. Macrophages, through scavenger receptors, recognize and abnormally phagocytize oxidized low-density lipoprotein (ox-LDL) in the blood, transforming into cholesteryl ester-rich foam cells. This process is not only a core mechanism for the formation of atherosclerotic plaques but also directly drives the progression of atherosclerosis by triggering a persistent inflammatory response. Recent studies have shown that directly inhibiting abnormal lipid accumulation and foam cell formation in macrophages can address the development of atherosclerosis at its source. However, existing clinical treatments (such as statins and PCSK9 inhibitors) primarily slow disease progression by regulating blood lipid levels, but fail to directly and specifically block macrophage foam cell formation, a key pathological component, and have limited efficacy in reversing plaques. Therefore, developing novel therapeutic strategies targeting the mechanisms of macrophage lipid accumulation and foam cell formation has become an important approach to overcoming current clinical bottlenecks. This invention provides a novel target targeting the core mechanism of foam cell formation, offering new insights into the treatment of atherosclerosis and possessing significant clinical and socioeconomic significance. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a treatment plan targeting macrophage Angulin-1, which, by promoting the expression of Angulin-1 in macrophages, inhibits the transformation of macrophages into a phenotype (foam cell) that promotes atherosclerosis function, thereby intervening in the course of atherosclerosis.

[0004] In order to achieve the purpose of the present invention, the technical solution of the present invention is as follows: In the first aspect, the present invention provides the use of macrophage Angulin-1 gene and / or Angulin-1 protein in the preparation or screening of drugs for preventing, alleviating, assisting in the treatment or / and treatment of atherosclerosis and related cardiovascular diseases.

[0005] In a second aspect, the present invention provides the use of macrophage Angulin-1 gene and / or Angulin-1 protein promoters in preparing drugs for preventing, alleviating, assisting in the treatment or / and treatment of atherosclerosis and related cardiovascular diseases, and in enhancing the expression and / or activity and / or function of the Angulin-1 gene and / or Angulin-1 protein.

[0006] Furthermore, the promoter targets and regulates the expression of Angulin-1 gene and / or Angulin-1 protein by regulating signal pathways.

[0007] Furthermore, the promoter includes: a recombinant expression vector containing the Angulin-1 gene or a gene fragment with more than 90% homology to the Angulin-1 gene, at least one of a polypeptide, protein, nucleic acid and nucleic acid aptamer, natural active substance, biological preparation, and compound capable of upregulating the expression of the Angulin-1 gene and / or Angulin-1 protein.

[0008] Furthermore, the recombinant expression vector includes any one of a vector-based eukaryotic expression plasmid, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, LNP liposome, an expression vector constructed by microinjection technology, a gene editing system element or a homologous recombination vector that specifically targets the Angulin-1 gene.

[0009] In a third aspect, the present invention provides a drug comprising the Angulin-1 gene and / or Angulin-1 protein described in claim 1, or at least one of the expression promoters of the above-mentioned Angulin-1 gene and / or Angulin-1 protein, as the main functional component of the drug.

[0010] Furthermore, the drug also includes pharmaceutically acceptable excipients, which include at least one of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants. The drug is in the form of any of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants, and sprays.

[0011] Furthermore, the mass percentage of the functional ingredient in the medicine is 1%-99%.

[0012] In a fourth aspect, the present invention provides the use of a reagent for detecting the expression level of Angulin-1 gene and / or protein in macrophages in the preparation of a product for auxiliary diagnosis, screening or evaluation of atherosclerosis and related cardiovascular diseases.

[0013] Furthermore, the product includes any one of a chip, a diagnostic reagent, and a diagnostic kit.

[0014] Furthermore, the test sample of the product is any one of cells, serum or whole blood.

[0015] Furthermore, the product contains antibodies that specifically bind to Angulin-1 protein or a peptide segment of Angulin-1 protein, or contains primers that specifically amplify Angulin-1 gene, or contains probes that specifically detect Angulin-1 gene.

[0016] The beneficial effects of the present invention are:

[0017] (1) The present invention first discovered that by regulating the expression level of Angulin-1 in macrophages, the uptake of oxidized low-density lipoprotein (ox-LDL) by macrophages can be effectively inhibited, the formation of foam cells can be blocked, and the plaque area and necrotic core area in the aorta and aortic root can be reduced. The present invention experimentally confirmed that the loss of macrophage Angulin-1 leads to the LDLR of high cholesterol diet. - / - The plaque area and necrotic core area of ​​mice increased significantly, promoting the occurrence and development of atherosclerosis; the bone marrow transplantation model revealed that the deficiency of Angulin-1 in macrophages increased LDLR in patients with a high cholesterol diet. - / - The formation and development of atherosclerotic plaques in mice; and the LDLR - / - After the expression level of Angulin-1 in macrophages of mice returned to normal, the plaque area and necrotic core area of ​​their aorta were significantly reduced, effectively inhibiting the occurrence and development of atherosclerosis; cell experiments confirmed that promoting the expression of Angulin-1 in macrophages can inhibit the macrophages from taking up oxidized low-density lipoprotein and inhibiting their transformation into foam cells; while reducing the expression of Angulin-1 in macrophages will promote the macrophages to take up oxidized low-density lipoprotein and transform into foam cells, promoting the occurrence and development of atherosclerosis. Compared with traditional treatment methods that only target superficial symptoms such as dyslipidemia or vascular stenosis, the present invention blocks the progression of the disease from the cell biology and molecular biology levels, reduces the risk of adverse cardiovascular events, significantly improves the treatment effect of the disease, and provides a more efficient solution for clinical treatment.

[0018] (2) The present invention provides a variety of intervention strategies to promote the expression of macrophage Angulin-1, which can be flexibly selected based on the patient's individual genetic background, the stage of disease progression, and the actual clinical medical conditions. For example, for patients with critical conditions, viral vector-mediated gene transfection technology can be used to quickly achieve efficient expression of the Angulin-1 gene; for patients seeking low toxic side effects, functional expression agonists of macrophage Angulin-1 can be used to target and regulate related signaling pathways. This diversified treatment strategy significantly improves the clinical adaptability of the treatment plan and provides an effective means to achieve personalized and precise treatment of atherosclerosis.

[0019] (3) This invention uses the Angulin-1 gene and its encoded protein in macrophages as a novel drug target, opening up a new direction for the development of anti-atherosclerotic drugs. The functional expression agonists developed based on this target have the significant advantages of high specificity and low toxicity and side effects. Combined with a variety of dosage forms such as tablets, capsules, and injections, personalized dosing regimens can be formulated based on the patient's medication habits, helping to improve patient medication compliance and providing more high-quality options for the treatment of atherosclerosis.

[0020] (4) The present invention provides a chip, diagnostic reagent, or kit for the preparation of auxiliary diagnosis of atherosclerosis using the macrophage Angulin-1 gene or protein as a target. This discovery helps to achieve early diagnosis of the disease, allowing patients to receive targeted treatment in the early stages of the disease, thereby effectively delaying the progression of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figures 1-4 The results of the diagram show the effect of macrophage deficiency of Angulin-1 on oxidized low-density lipoprotein uptake and foam cell formation, specifically: Figure 1 A is an immunofluorescence image of dil-ox-LDL uptake by bone marrow-derived macrophages with normal and Angulin-1 deficiency, and B is a quantitative image of dil fluorescence intensity in the culture supernatant and cell lysate of bone marrow-derived macrophages with normal and Angulin-1 deficiency; Figure 2 A is the immunofluorescence image of dil-ox-LDL uptake by peritoneal macrophages with normal and Angulin-1 deficiency, and B is the quantitative image of dil fluorescence intensity in the culture supernatant and cell lysate of peritoneal macrophages with normal and Angulin-1 deficiency; Figure 3 Oil red O staining of ox-LDL uptake by bone marrow-derived macrophages with normal and Angulin-1 deficiency; Figure 4 Oil red O staining of ox-LDL uptake by peritoneal macrophages with normal and Angulin-1 deficiency;

[0022] Figure 5-Figure 8 The results of the effect of Angulin-1 deficiency in macrophages on the expression of receptors related to cholesterol metabolism are as follows: Figure 5 The results of qRT-PCR detection of the expression levels of cholesterol metabolism-related receptors in bone marrow-derived macrophages with normal and Angulin-1 deficiency are shown; Figure 6 Western blot analysis of LOX-1 expression in normal and Angulin-1-deficient bone marrow-derived macrophages; Figure 7 The results of qRT-PCR detection of the expression levels of cholesterol metabolism-related receptors in peritoneal macrophages with normal and Angulin-1 deficiency are shown; Figure 8 Western blot analysis of LOX-1 expression in normal and Angulin-1-deficient peritoneal macrophages;

[0023] Figures 9-12 The results of the blockade of LOX-1 neutralizing antibodies on the uptake of oxidized low-density lipoprotein and foam cell formation in macrophages lacking Angulin-1 are shown in the figure below. Figure 9 A is an immunofluorescence image of dil-ox-LDL uptake by bone marrow-derived macrophages lacking Angulin-1 after being treated with control antibody IgG and LOX-1 neutralizing antibody; B is a quantitative image of dil fluorescence intensity in the culture supernatant and cell lysate of bone marrow-derived macrophages lacking Angulin-1 after being treated with control antibody IgG and LOX-1 neutralizing antibody; Figure 10 A is an immunofluorescence image of dil-ox-LDL uptake by peritoneal macrophages lacking Angulin-1 after being treated with control antibody IgG and LOX-1 neutralizing antibody; B is a quantitative image of dil fluorescence intensity in the culture supernatant and cell lysate of peritoneal macrophages lacking Angulin-1 after being treated with control antibody IgG and LOX-1 neutralizing antibody; Figure 11 Oil Red O staining of ox-LDL taken up by bone marrow-derived macrophages lacking Angulin-1 after being treated with control antibody IgG and LOX-1 neutralizing antibody; Figure 12 The figure is Oil Red O staining of ox-LDL uptake by peritoneal macrophages lacking Angulin-1 after being treated with control antibody IgG and LOX-1 neutralizing antibody;

[0024] Figures 13-18 LDLR in macrophages lacking Angulin-1 - / - (Angulin-1 MKO LDLR - / - ) LDLR normally expressed in mice and Angulin-1 in macrophages - / - (Angulin-1 Ctrl LDLR - / - ) The test results of mice fed a high-cholesterol diet for 30 weeks are as follows: Figure 13 Angulin-1 Ctrl LDLR - / - Mice and Angulin-1 MKO LDLR - / - Diagram of mouse generation and modeling; Figure 14 Angulin-1 Ctrl LDLR - / - Mice and Angulin-1 MKO LDLR- / - Oil red staining of whole aorta of mice and quantitative results; Figure 15 Angulin-1 Ctrl LDLR - / - Mice and Angulin-1 MKO LDLR - / - Oil red staining images and quantitative results of the aortic root of mice; Figure 16 Angulin-1 Ctrl LDLR - / - Mice and Angulin-1 MKO LDLR - / - HE staining and quantitative results of the aortic root of mice; Figure 17 Angulin-1 Ctrl LDLR - / - Mice and Angulin-1 MKO LDLR - / - Quantitative results of necrotic core in HE staining of mouse aortic root; Figure 18 Angulin-1 Ctrl LDLR - / - Mice and Angulin-1 MKO LDLR - / - Oil red staining of mouse peritoneal macrophages;

[0025] Figures 19-23 Angulin-1 Ctrl Mice and Angulin-1 MKO Bone marrow cells of mice transplanted into LDLR - / - Mouse chimera Angulin-1 Ctrl →LDLR - / - Mice and Angulin-1 MKO →LDLR - / - The test results of mice fed a high-cholesterol diet for 24 weeks are as follows: Figure 19 Angulin-1 Ctrl →LDLR - / - Mice and Angulin-1 MKO →LDLR - / - Schematic diagram of mouse generation and modeling; Figure 20 Angulin-1 Ctrl →LDLR - / - Mice and Angulin-1 MKO →LDLR - / - Oil red staining of whole aorta of mice and quantitative results; Figure 21 Angulin-1 Ctrl →LDLR - / -Mice and Angulin-1 MKO →LDLR - / - Oil red staining images and quantitative results of the aortic root of mice; Figure 22 Angulin-1 Ctrl →LDLR - / - Mice and Angulin-1 MKO →LDLR - / - Quantitative results of necrotic core in HE staining of mouse aortic root; Figure 23 Angulin-1 Ctrl →LDLR - / - Mice and Angulin-1 MKO →LDLR - / - Oil red staining of mouse peritoneal macrophages;

[0026] Figure 24-27 The results of the overexpression of Angulin-1 in macrophages on the uptake of oxidized low-density lipoprotein and the formation of foam cells are shown as follows: Figure 24 A is an immunofluorescence image of dil-ox-LDL uptake by normal and Angulin-1-overexpressing bone marrow-derived macrophages; B is a quantitative image of dil fluorescence intensity in the culture supernatant and cell lysate of normal and Angulin-1-overexpressing bone marrow-derived macrophages; Figure 25 A is an immunofluorescence image of dil-ox-LDL uptake by normal and Angulin-1-overexpressing peritoneal macrophages; B is a quantitative image of dil fluorescence intensity in the culture supernatant and cell lysate of normal and Angulin-1-overexpressing peritoneal macrophages; Figure 26 Oil red O staining of ox-LDL uptake by normal and Angulin-1 overexpressing bone marrow-derived macrophages; Figure 27 Oil red O staining of ox-LDL uptake by normal and Angulin-1 overexpressing peritoneal macrophages;

[0027] Figures 28-31 Angulin-1 MKO Mice and Angulin-1 Ctrl Angulin-1 MKO LDLR - / - Mouse chimera Angulin-1 MKO →Angulin-1 MKO LDLR - / - Mice and Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - The test results of mice fed a high-cholesterol diet for 24 weeks are as follows: Figure 28 Angulin-1 MKO →Angulin-1 MKO LDLR - / - Mice and Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - Schematic diagram of mouse generation and modeling; Figure 29 Angulin-1 MKO →Angulin-1 MKO LDLR - / - Mice and Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - Oil red staining of whole aorta of mice and quantitative results; Figure 30 Angulin-1 MKO →Angulin-1 MKO LDLR - / - Mice and Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - Oil red staining images and quantitative results of the aortic root of mice; Figure 31 Angulin-1 MKO →Angulin-1 MKO LDLR - / - Mice and Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - Oil red staining of mouse peritoneal macrophages;

[0028] Figure 32 This is an experimental verification diagram provided by the present invention that reveals that macrophage Angulin-1 is used as a target to intervene in the course of atherosclerosis. DETAILED DESCRIPTION

[0029] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features therein can be combined with each other.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all the embodiments.

[0031] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0032] In this specification, the expressions “comprise” and “include” include concepts such as “comprise”, “include”, “consist essentially of” and “consist only of”.

[0033] "Homology" of a nucleotide sequence refers to the degree of identity of two or more nucleotide sequences that can be compared relative to each other's nucleotide sequences. Therefore, the higher the identity of two nucleotide sequences, the higher the identity or similarity of these sequences. The level of identity of a nucleotide sequence can be determined, for example, using FASTA, a tool for sequence analysis, using default parameters. Alternatively, it can be determined using the BLAST algorithm based on Karlin and Altschul. A program called BLASTX based on such a BLAST algorithm has been developed. The specific methods of these analytical methods are well known and can be found on the website (http: / / www.ncbi.nlm.nih.gov / ) of the National Center of Biotechnology Information (NCBI).

[0034] In this specification, the abbreviation "Angulin-1" refers to the protein.

[0035] The present invention relates to a recombinant expression vector comprising an angulin-1 gene expression regulatory region and a gene configured for expression controlled by the region. A "recombinant expression vector" refers to a polynucleotide capable of expressing the gene contained in the expression cassette in a cell (e.g., a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell). The type of vector is not particularly limited and may include any of a vector-based eukaryotic expression plasmid, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, LNP liposome, an expression vector constructed by microinjection technology, a gene editing system component, or a homologous recombination vector that specifically targets the angulin-1 gene.

[0036] The vector of the present invention can also be contained in cells. Such cells (hereinafter referred to as "cells of the present invention") are also included in the present invention. In the cells of the present invention, the vector of the present invention can also exist outside the genome, or can also exist in a state of being incorporated into the genome. There is no particular limitation on the biological species of origin of the cell, and examples thereof include: various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. In addition, there is no particular limitation on the type of the cell, and examples thereof include: cells derived from various tissues or having various properties, such as blood cells, hematopoietic stem cells / precursor cells, fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, etc.

[0037] In the present invention, the "Angulin-1 gene" is not particularly limited, and examples thereof include genes derived from various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer.

[0038] The "Angulin-1 protein" of the present invention can be a naturally occurring protein or can be produced as a recombinant protein using genetic recombination techniques. Naturally occurring proteins can be produced from an extract of macrophages expressing the angulin-1 protein by affinity chromatography using antibodies against the angulin-1 protein. Of course, recombinant proteins can be produced by culturing cells transformed with DNA encoding the angulin-1 protein.

[0039] The angulin-1 gene expression promoters of the present invention are one or more combinations of compounds, polypeptides, proteins, nucleic acids and nucleic acid aptamers, natural active substances, and biological agents that can upregulate angulin-1 gene and / or protein expression. Angulin-1 protein promoters include substances that target and regulate angulin-1 gene and / or protein expression and / or activity by modulating signaling pathways. The presence or absence of promoter activity can be determined according to known methods or the methods described in the Examples.

[0040] In specific implementations, promoting the expression of Angulin-1 gene and / or protein in macrophages can be achieved through a variety of technical pathways:

[0041] (1) Gene transfection technology: Construct a eukaryotic expression plasmid vector targeting the Angulin-1 gene, use liposomes and other transfection reagents, and introduce the vector into the macrophage culture system (37°C, 5% CO2 environment) according to standard operating procedures to achieve intracellular expression of the Angulin-1 gene.

[0042] (2) Viral vector-mediated delivery: Gene delivery is performed using viral vector systems such as adenovirus, adeno-associated virus, and lentivirus. Taking lentivirus as an example, the process of constructing an Angulin-1 gene vector, packaging 293T cells, and infecting macrophages must be completed in sequence to achieve stable integration and expression of the Angulin-1 gene. Special vectors such as baculovirus and herpes virus can be used for differentiated operations based on their infection characteristics.

[0043] (3) Novel delivery systems and gene editing: Lipid nanoparticles (LNPs), gene editing system components (such as the CRISPR / Cas9 system), and homologous recombination vectors are all optional technologies. When using the CRISPR / Cas9 system, specific sgRNAs must be designed for the regulatory region of the Angulin-1 gene and co-transfected with the Cas9 protein or expression vector to achieve enhanced gene expression. Microinjection technology directly delivers Angulin-1 gene nucleic acid solutions or Angulin-1 protein agonists into macrophages to rapidly increase Angulin-1 expression levels.

[0044] (4) Signal pathway regulation: Screening for substances that target and regulate the expression and / or activity of Angulin-1 gene and / or protein, activating the signal pathway that promotes Angulin-1 expression, or inhibiting the signal pathway that negatively regulates the expression of Angulin-1 gene and / or protein, to achieve upregulation of expression at the transcriptional and translational levels.

[0045] Furthermore, substances that enhance the expression and / or function of Angulin-1 gene or protein can be used in the following aspects:

[0046] (1) Preparation of products for the prevention and / or treatment of atherosclerosis;

[0047] (2) Preparation of products for inhibiting macrophage uptake of oxidized low-density lipoprotein;

[0048] (3) Preparation of products for inhibiting macrophage foaming.

[0049] By making these substances into suitable dosage forms, such as oral dosage forms such as tablets and capsules, or parenteral dosage forms such as injections, the prevention and treatment of atherosclerosis can be exerted from different angles.

[0050] The animal experimental data and clinical research results corroborate each other, providing sufficient theoretical basis and practical support for the above-mentioned applications, and confirming the effectiveness and feasibility of enhancing Angulin-1 expression and function in the prevention and treatment of atherosclerosis.

[0051] The pharmaceutical compositions and / or adjuvant pharmaceutical compositions provided herein comprise a functional expression agonist capable of promoting the expression of the Angulin-1 gene and / or protein in macrophages, as well as pharmaceutically acceptable active ingredients and excipients. The active ingredients capable of promoting the expression of the Angulin-1 gene and / or protein in macrophages include nucleic acid molecules encoding the Angulin-1 gene, small molecule compounds that upregulate Angulin-1 expression, macromolecular bioactive substances (such as peptides, proteins, and nucleic acid aptamers), and biological agents, and can be formulated appropriately based on specific needs.

[0052] In the present invention, the term "pharmaceutically acceptable" refers to any molecular entity or composition that does not produce an adverse, allergic, or other inappropriate or unwanted reaction when administered to a subject. As used herein, the term "pharmaceutically acceptable composition" is synonymous with "pharmaceutical composition" and refers to a therapeutically effective concentration of an active ingredient (e.g., any therapeutic compound disclosed herein). The pharmaceutical compositions disclosed herein can be used for medical and veterinary applications. The pharmaceutical compositions can be administered to a subject alone or in combination with other supplementary active ingredients, agents, drugs, or hormones.

[0053] The pharmaceutical compositions disclosed herein may optionally include a pharmaceutically acceptable carrier to facilitate processing of the active ingredient into a pharmaceutically acceptable composition. As used herein, the term "pharmacologically acceptable carrier" is synonymous with "pharmaceutical carrier" and refers to any carrier that has substantially no long-term or permanent adverse effects when administered, and includes terms such as "pharmacologically acceptable carriers, stabilizers, diluents, additives, adjuvants, or excipients." Such carriers are typically mixed with the active compound, or allow for dilution or encapsulation of the active compound, and may be solid, semisolid, or liquid agents. It will be appreciated that the active ingredient may be soluble or may be delivered as a suspension in a desired carrier or diluent. Any of a variety of pharmaceutically acceptable carriers may be used, including, but not limited to, aqueous media such as water, saline, glycine, hyaluronic acid, and the like; solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption-delaying agents; or any other inactive ingredients. The pharmacologically acceptable carrier may be selected based on the intended mode of administration. Except insofar as any pharmacologically acceptable carrier is incompatible with the active ingredient, its use in a pharmaceutically acceptable composition is contemplated.

[0054] The pharmaceutical compositions disclosed herein may optionally contain, but are not limited to, other pharmaceutically acceptable components (or pharmaceutical components), including but not limited to buffers, preservatives, isotonicity regulators, salts, antioxidants, osmotic pressure regulating agents, physiological substances, pharmacological substances, fillers, emulsifiers, wetting agents, sweeteners or flavoring agents, etc. Various buffers and means for adjusting pH can be used to prepare the pharmaceutical compositions disclosed herein, provided that the resulting formulation is pharmaceutically acceptable.

[0055] In addition, the present invention also provides the use of a reagent for detecting the expression level of Angulin-1 gene and / or protein in macrophages in the preparation of a product for auxiliary diagnosis, screening or evaluation of atherosclerosis and related cardiovascular diseases.

[0056] The method of preventing and / or treating atherosclerosis is achieved by promoting the expression of the Angulin-1 gene and / or protein in macrophages. The occurrence and development of atherosclerosis is closely related to the uptake of oxidized low-density lipoprotein by macrophages and their conversion into foam cells. It should be noted that the specific indications for "atherosclerosis and related cardiovascular diseases" are as follows:

[0057] (1) Core indications

[0058] It is divided into primary lesions and affected vascular diseases. The former includes patients with confirmed atherosclerosis who have been confirmed by imaging to have lipid deposition in the arterial intima and plaque formation (such as coronary CT angiography showing lumen stenosis ≥50% or carotid ultrasound showing carotid intima-media thickness thickening ≥1.5mm with plaques), and patients with unstable plaque status who have been confirmed by intravascular ultrasound or optical coherence tomography (OCT) to have incomplete plaque fibrous cap, exposed lipid pool or intraplaque hemorrhage; the latter involves stable coronary heart disease in the coronary artery system (meeting CCS grade of angina pectoris I-IV and coronary angiography showing vascular stenosis ≥70%) and unstable coronary syndrome (including unstable angina, non-ST segment elevation and ST The patients were diagnosed with cerebral infarction (within 72 hours of onset), cerebral infarction caused by atherosclerosis confirmed by imaging within 72 hours of onset of the cerebrovascular system, transient ischemic attack with plaques in the carotid artery or vertebrobasilar artery system, lower limb artery disease of Fontaine stage (clinical staging of lower limb arteriosclerosis obliterans) II or above, and renal artery atherosclerotic hypertension with renal artery stenosis ≥70% and difficult-to-control blood pressure, and other involved vascular diseases.

[0059] (2) Indications for complications

[0060] These include chronic heart failure with NYHA class II-IV and left ventricular ejection fraction (LVEF) ≤40% caused by atherosclerotic heart disease, heart failure with left ventricular ejection fraction (LVEF) ≤50% after acute myocardial infarction, ventricular arrhythmias such as frequent ventricular premature beats ≥1000 times / 24 hours or non-sustained ventricular tachycardia, and severe atrioventricular block requiring pacemaker treatment after myocardial infarction, as well as acute pulmonary embolism caused by plaque rupture and aortic aneurysm with a diameter ≥4 cm or a growth of ≥0.5 cm in half a year.

[0061] (3) Preventive indications

[0062] It is intended for primary prevention in high-risk groups with a 10-year ASCVD risk ≥10% and ≥3 risk factors such as hypertension and diabetes, as well as for secondary prevention in patients who have already suffered myocardial infarction, stroke or peripheral arterial revascularization.

[0063] (4) Combined indications

[0064] It is suitable for patients with metabolic syndrome (meeting ≥3 abnormalities such as waist circumference, triglycerides, etc.) or chronic kidney disease (eGFR < 60 ml / min / 1.73 m² and positive urine protein).

[0065] This product accurately detects Angulin-1 gene or protein expression levels in samples through a variety of detection methods, such as qRT-PCR, in situ hybridization, Northern Blot, Western Blot, microarrays, high-throughput sequencing platforms, immunohistochemistry, or enzyme-linked immunosorbent assay (ELISA). The product contains antibodies (including monoclonal antibodies, polyclonal antibodies, or single-domain antibodies) that specifically bind to Angulin-1 protein or peptide fragments of Angulin-1 protein, primers that specifically amplify the Angulin-1 gene, or probes that specifically detect the Angulin-1 gene. The test sample can be cells, serum, or whole blood, and the product can be in the form of a microarray, diagnostic reagent, or kit. This type of testing product can provide a basis for the early diagnosis and disease assessment of atherosclerosis, helping clinicians develop personalized diagnosis and treatment plans and improving the efficiency and quality of disease diagnosis and treatment.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0067] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0068] Example 1

[0069] Effects of Angulin-1 gene deletion in macrophages on ox-LDL uptake and foam cell formation.

[0070] 1. Experimental Animals

[0071] (1) Angulin-1 loxP / loxP mice

[0072] Angulin-1 loxP / loxP The mice were constructed by the inventors using CRISPR Cas9 technology. The construction process is as follows:

[0073] In vitro, a gene sequence containing loxP sites was inserted on both sides of the first exon of the Angulin-1 gene. The recombinant gene sequence was then transferred into embryonic stem cells of C57BL / 6J mice through electroporation. The embryonic stem cells were then reimplanted into the uterus of pseudopregnant mice, allowing them to develop into a complete embryo and produce a transgenic mouse. The transgenic mouse was then hybridized with C57BL / 6J mice (the purpose of the hybridization was to remove the Neo transgene) to obtain Angulin-1. loxP / - Mice, then Angulin-1 loxP / - Mice are self-fertilized and eventually produce Angulin-1 loxP / loxP mouse.

[0074] (2) LysM-Cre mice

[0075] LysM-Cre mice expressing macrophage-specific Cre recombinase were purchased from Jackson Laboratory and are also known as Lyz2-Cre mice.

[0076] (3) Angulin-1 loxP / loxP LysM-Cre + / - Macrophage Angulin-1 conditional knockout mice (Angulin-1 MKO ) and Angulin-1 loxP / loxP LysM-Cre - / - Wild-type mice (Angulin-1 Ctrl )

[0077] Angulin-1 MKO Mice and Angulin-1 Ctrl The mice were all raised by the inventors by making Angulin-1 loxP / loxP The mice were hybridized with LysM-Cre mice. The acquisition process is as follows:

[0078] Angulin-1 loxP / loxP Mice and heterozygous mice expressing cre recombinase specifically in macrophages (LysM-cre + / -) and the genotype Angulin-1 loxP / - LysM-cre + / - The offspring mice and Angulin-1 loxP / loxP Backcross mice, according to Mendel's law of inheritance, the genotype of their offspring may be Angulin-1 loxp / - LysM-cre + / - 、Angulin-1 loxp / loxp LysM-cre + / - 、Angulin-1 loxp / loxp LysM-cre - / - and Angulin-1 loxp / - LysM-cre - / - Among them, Angulin-1 loxp / loxp LysM-cre + / - Macrophage-specific Angulin-1 gene knockout mice (Angulin-1 MKO ), Angulin-1 loxp / loxp LysM-cre - / - As a control group (Angulin-1 Ctrl ).

[0079] 2. Cell Processing

[0080] (1) Extraction of peritoneal macrophages

[0081] To Angulin-1 Ctrl Mice and Angulin-1 MKO Mice were intraperitoneally injected with 4% thioglycolate medium (3 mL / mouse). Seventy-two hours later, the mice were sacrificed, and the peritoneal cavity was lavaged with 20 mL of PBS. The lavage fluid was collected and centrifuged (500 × g, 3 min). After resuspending in 1640 medium supplemented with 10% FBS, the cells were incubated at 37°C in a 5% CO2 incubator. Non-adherent cells were discarded after 1 hour to obtain peritoneal macrophages.

[0082] (2) Extraction and induction of bone marrow-derived macrophages

[0083] Extract Angulin-1 Ctrl Mice and Angulin-1 MKO Mouse bone marrow cells were lysed with red blood cell lysis buffer for 2 minutes, resuspended in 1640 medium containing 10% FBS, and inoculated into culture dishes for 6 days. Fresh medium was replaced every 3 days. M-CSF cytokine was added on the first and third days of culture to obtain bone marrow-derived macrophages.

[0084] 3. ox-LDL uptake experiment

[0085] Mouse peritoneal macrophages were extracted and plated in 24-well plates at a confluence of approximately 90%. 50 μg / mL Dil-ox-LDL was added and incubated for 4 h. The culture medium was collected and the fluorescence intensity was measured using a multifunctional fluorescence microplate reader to determine the amount of Dil-ox-LDL remaining in the supernatant. The cells were lysed, and the fluorescence intensity of the lysate was measured to determine the amount of Dil-ox-LDL taken up. Protein concentration was determined for calibration.

[0086] 4. Foam cell detection: Macrophage Oil Red O staining

[0087] Extract Angulin-1 Ctrl Mice and Angulin-1 MKO Mouse peritoneal macrophages or bone marrow-derived macrophages were plated in 24-well plates pre-covered with coverslips to a confluence of approximately 90%. 50 μg / mL ox-LDL was added and incubated for 24 h. The cells were washed three times with PBS and fixed with tissue cell fixative for 30 min at room temperature. The cells were washed three times with PBS and stained with Oil Red O for 40 min. The cells were washed three times with PBS and stained with hematoxylin for 20 s. The cells were bluish-red in tap water for 5 min and then soaked in distilled water for 5 min. After sealing and air-drying, the cells were photographed and analyzed using an optical microscope. Cells with >10 lipid droplets were defined as foam cells, and the proportion of foam cells was calculated.

[0088] 5. Data Analysis

[0089] Prism 8 software was used to analyze the experimental data. Unpaired t-test was used and the data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

[0090] 6. Experimental Results

[0091] Macrophage-specific knockout of Angulin-1 significantly promoted the uptake of ox-LDL (e.g. Figure 1 and Figure 2 ) and foam cell formation (as shown in Figure 3 and Figure 4 As shown in Figure 3 ). It can be seen that Angulin-1 deficiency promotes the uptake of dil-ox-LDL and the formation of foam cells, indicating that Angulin-1 may play an important role in the occurrence and development of atherosclerosis.

[0092] Example 2

[0093] Angulin-1 deficiency in macrophages mediates foam cell formation through LOX-1.

[0094] 1. Cell processing

[0095] (1) Extraction of peritoneal macrophages

[0096] Same as Example 1.

[0097] (2) Extraction of macrophages induced by bone marrow cells

[0098] Same as Example 1.

[0099] 2. Antibody blocking experiment

[0100] (1) Group: Angulin-1 MKO Primary peritoneal macrophages or bone marrow-derived macrophages of mice were randomly divided into two groups: the control group, which was treated with IgG (Invitrogen, USA), and the anti-LOX-1 group, which was treated with LOX-1 neutralizing antibody (R&D Systems, USA).

[0101] (2) Blocking experiment

[0102] ① Discard the culture medium: Carefully aspirate and discard the original culture medium in the 6-well plate containing primary peritoneal macrophages or bone marrow-derived macrophages to avoid blowing away the adherent cells.

[0103] ②PBS washing: Add 2 mL of sterile PBS to each well, gently shake the culture plate, let it stand at room temperature for 2-3 minutes, then aspirate and discard the PBS. Repeat the washing twice to remove residual culture medium components.

[0104] ③ Add antibodies: Add 1 mL of diluted LOX-1 neutralizing antibody solution to each well, ensuring that the solution evenly covers the cell surface. Place the culture plate in a CO2 incubator and incubate at 37°C for 2-4 hours. Avoid moving the culture plate frequently during this period.

[0105] 3. RNA Extraction and Real-time Quantitative PCR

[0106] Cells were homogenized in Trizol (Ambion, USA) to obtain total RNA. RNA was reverse transcribed using a reverse transcription system kit according to the manufacturer's instructions (Takara, Japan). Quantitative PCR was then performed using a ThermoFisher QS3 PCR instrument according to the instructions provided with the SYBR Green PCR Master Mix (ABI, USA) kit. The internal reference gene was β-actin, and the expression of ΔCt = Ct was used. gene -Ct β-actin Calculate the relative expression level of each target gene mRNA and use 2 -ΔCt Value representation.

[0107] 4. Western blot

[0108] (1) Sample preparation

[0109] Cultured macrophages were harvested, rinsed with PBS, and then trypsinized. The reaction was terminated by adding serum-containing medium and the cells were collected by centrifugation. RIPA lysis buffer containing inhibitors was added and the cells were lysed on ice for 30 minutes with vortexing. The supernatant was collected by centrifugation and the protein concentration was determined using a BCA kit.

[0110] (2) Protein separation

[0111] Prepare separating gel and stacking gel according to the molecular weight of the target protein. Mix the protein sample with SDS loading buffer and boil for denaturation. Add 20-30 μg of sample and protein marker to each well. First, electrophorese at 80V until the protein flows into the separating gel. Then adjust to 120-150V until the bromophenol blue reaches the bottom of the gel.

[0112] (3) Transfer

[0113] Activate and equilibrate the PVDF membrane. Assemble the transfer apparatus in the correct order, ensuring there are no bubbles. Transfer the membrane at a constant voltage of 100 V for 1 hour.

[0114] (4) Follow-up testing

[0115] Block the membrane with 5% skim milk or BSA for 1 hour. After washing with TBST, incubate with the primary antibody overnight at 4°C and then with the secondary antibody for 1-2 hours at room temperature. Wash thoroughly with TBST three times after each incubation. Prepare ECL luminescent solution for color development, expose the membrane on a chemiluminescence imager, analyze the grayscale value of the bands using Image J, and calculate the relative expression level of the target protein.

[0116] 5. ox-LDL uptake experiment

[0117] Same as Example 1.

[0118] 6. Foam cell detection: Macrophage Oil Red O staining

[0119] Same as Example 1.

[0120] 7. Data Analysis

[0121] Prism 8 software was used to analyze the experimental data. Unpaired t-test was used and the data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

[0122] 8. Experimental Results

[0123] like Figure 5-Figure 8As shown in the results, it was found that macrophage Angulin-1 deficiency could significantly promote the upregulation of LOX-1 expression without affecting the expression of other oxidized low-density lipoprotein uptake receptors and cholesterol transport proteins ABCA1 and ABCG1.

[0124] like Figures 9-12 As shown, by blocking LOX-1 on the surface of angulin-1-deficient macrophages using a LOX-1 neutralizing antibody, it was found that the uptake of ox-LDL and the transformation of macrophages into foam cells were effectively inhibited.

[0125] These results indicate that the loss of macrophage Angulin-1 upregulates the expression of LOX-1, thereby promoting the formation of foam cells.

[0126] Example 3

[0127] Macrophage Angulin-1 deficiency promotes the development of atherosclerosis.

[0128] 1. Experimental Animals

[0129] (1) LDLR - / - mice

[0130] LDLR - / - Mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0131] (2) Angulin-1 double knockout mice MKO LDLR - / - Build

[0132] LDLR - / - Mice and Angulin-1 loxp / loxp LysM-cre + / - Crossing was performed to obtain Angulin-1 of the F1 generation loxp / - LysM-cre + / - LDLR + / - and LDLR - / - Mouse backcross, Angulin-1 loxp / - LysM-cre - / - LDLR + / - Angulin-1 loxp / loxp LysM-cre + / - Backcrossing mice; obtaining Angulin-1 in the F2 generation loxp / - LysM-cre + / - LDLR - / - Angulin-1 loxp / - LysM-cre + / - LDLR + / -or Angulin-1 loxp / - LysM-cre - / - LDLR + / - Hybrid, Angulin-1 loxp / - LysM-cre - / - LDLR - / - Angulin-1 loxp / loxp LysM-cre + / - LDLR + / - Hybridization; finally obtain F3 generation Angulin-1 loxp / loxp LysM-cre + / - LDLR - / - (Angulin-1 MKO LDLR - / - ) mice and Angulin-1 loxp / loxp LysM-cre - / - LDLR - / - (Angulin-1 Ctrl LDLR - / - ) mice.

[0133] 2. Modeling method

[0134] Angulin-1 Ctrl LDLR - / - Mice and Angulin-1 MKO LDLR - / - Mice were fed a high cholesterol diet (containing 1.25% cholesterol and 20% fat) and euthanized after 30 weeks. Peritoneal macrophages and aorta were collected for subsequent testing (e.g. Figure 13 shown).

[0135] 3. Extraction of peritoneal macrophages

[0136] Same as Example 1.

[0137] 4. Plaque area and stability detection

[0138] (1) Detection of whole aortic plaque area - Oil Red O staining method

[0139] The whole mouse aorta was placed under a stereomicroscope. The tissue around the aorta was peeled off with fine forceps and placed in a 24-well plate. The aorta was fixed with tissue cell fixative at 4°C for 24 h. The fixative was discarded and the aorta was stained with Oil Red O solution for 1 h. The floating color was washed off with 70% ethanol and the aorta was photographed with a stereomicroscope. The red color indicated atherosclerotic plaques. The plaque area and proportion were calculated.

[0140] (2) Detection of aortic root plaque area—Oil Red O staining

[0141] The aortic root was removed, dehydrated in 30% sucrose solution overnight, embedded in OCT, and cut into 5 μm thickness using a freezing microtome. The sections were stained with oil red for 1 h, washed with 60% isopropanol to remove the floating color, and photographed under a microscope to calculate the plaque size and area.

[0142] (3) Detection of necrotic core of aortic root - HE staining method

[0143] The aortic root sections were immersed in PBS for 30 minutes to fully remove OCT, then immersed in hematoxylin and stained for 10 minutes, blueed in tap water for 5 minutes, and immersed in distilled water for 5 minutes; immersed in eosin staining solution for 5 minutes; dehydrated and transparent by immersing in different gradient ethanol and xylene in sequence; sealed with neutral resin; dried, and photographed with an optical microscope to analyze indicators such as plaque morphology and necrotic core area.

[0144] 5. Data Analysis

[0145] Prism 8 software was used to analyze the experimental data. Unpaired t-test was used and the data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

[0146] 6. Experimental Results

[0147] Compared with the control group, Angulin-1 Ctrl LDLR - / - Compared with mice, we found that Angulin-1 MKO LDLR - / - The whole aorta of mice developed obvious atherosclerotic plaques, which were stained red by Oil Red O, while the areas without plaques were transparent. The area stained with Oil Red O and the area of ​​the whole aorta were measured using ImageJ and the ratio was calculated. Unpaired t-test analysis showed that Angulin-1 MKO LDLR - / - The total aorta plaque area ratio of group mice was significantly greater than that of the control group (e.g. Figure 14 shown).

[0148] Angulin-1 MKO LDLR - / - Mouse aortic root shows more than Angulin-1 Ctrl LDLR - / - The larger plaque area in mice (e.g. Figure 15 Further analysis of aortic root plaques by HE staining showed that Angulin-1 MKO LDLR - / - The atherosclerotic lesion area and necrotic core area of ​​mice were significantly increased (e.g. Figure 16 and Figure 17 shown).

[0149] A high cholesterol diet leads to angulin-1 Ctrl LDLR - / - About 10% of peritoneal macrophages in mice differentiate into foam cells (e.g. Figure 18 Angulin-1 MKO LDLR - / - The percentage of differentiated foam cells in mice increased to 34% (e.g. Figure 18 Figure 3 (shown), showing that mice lacking Angulin-1 in macrophages exhibited significantly increased macrophage lipid accumulation and foam cell formation.

[0150] Example 4

[0151] Macrophage Angulin-1 deficiency promotes the development of atherosclerosis in bone marrow transplantation experiments.

[0152] 1. Experimental Animals

[0153] (1) LDLR - / - mice

[0154] Same as Example 3

[0155] (2) Bone marrow transplantation

[0156] ① Extraction of bone marrow cells from donor mice: Angulin-1 Ctrl Mice and Angulin-1 MKO After euthanasia, the femur and tibia were stripped, soaked in 75% alcohol for 5 min, and then soaked in PBS for 5 min. The bones were cut open at both ends and rinsed with sterile PBS. The collected cell suspension was filtered through a 40 μm filter and centrifuged at 500 g for 5 min. The supernatant was discarded, and red blood cell lysis buffer was added to the cell pellet for 3 min. The reaction was terminated by adding complete culture medium. The cells were centrifuged again, the supernatant was discarded, and the cells were resuspended in sterile PBS to a density of 1×10 7 cells / mL and place on ice until ready for use.

[0157] ② Preparation of bone marrow transplant recipient mice: One week before irradiation, the recipient LDLR - / - The mice drank sterile water containing gentamicin (0.4 g / L) and received X-ray irradiation (5.5 Gy, twice, 3 h apart). 6 h later, the donor mice were injected with Angulin-1 Ctrl Mice and Angulin-1 MKO Mouse) bone marrow cells 5×10 6 , forming chimeric mouse Angulin-1 Ctrl →LDLR - / - Mice and Angulin-1MKO →LDLR - / - Mice continued to drink gentamicin-containing water after irradiation, which was replaced with normal water after 2 weeks; and began to be fed high-cholesterol food 6 weeks after irradiation.

[0158] 2. Modeling method

[0159] Angulin-1 Ctrl →LDLR - / - Mice and Angulin-1 MKO →LDLR - / - Mice were fed a high cholesterol diet (containing 1.25% cholesterol and 20% fat) and euthanized after 24 weeks. Peritoneal macrophages and aorta were collected for subsequent testing (e.g. Figure 19 shown).

[0160] 3. Extraction of peritoneal macrophages

[0161] Same as Example 1.

[0162] 4. Plaque area and stability detection

[0163] Same as Example 2.

[0164] 5. Data Analysis

[0165] Prism 8 software was used to analyze the experimental data. Unpaired t-test was used and the data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

[0166] 6. Experimental Results

[0167] Angulin-1 Ctrl →LDLR - / - Compared with mice, Angulin-1 MKO →LDLR - / - The progression of atherosclerotic lesions in the entire aorta and aortic root of mice (e.g. Figure 20 and Figure 21 ), necrotic core area (as shown in Figure 22 ), the proportion of foam cells in peritoneal macrophages (as shown in Figure 23 This further demonstrates that macrophage Angulin-1 deficiency aggravates the development of atherosclerosis.

[0168] Example 5

[0169] To study the effect of overexpression of Angulin-1 in macrophages on the uptake of ox-LDL and the formation of foam cells.

[0170] 1. Experimental Animals

[0171] C57BL / 6J mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0172] 2. Cell Processing

[0173] (1) Extraction of peritoneal macrophages from wild-type C57BL / 6J mice

[0174] Same as Example 1.

[0175] (2) Extraction and induction of wild-type C57BL / 6J mouse bone marrow-derived macrophages

[0176] Same as Example 1.

[0177] (3) Construction of peritoneal macrophages overexpressing Angulin-1

[0178] Concentrated viral particles, pLVX-Angulin-1, were added to primary peritoneal macrophage cultures from wild-type C57BL / 6J mice at a multiplicity of infection (MOI) of 10. 8 μg / mL polybrene was also added, gently mixed, and the cells were incubated in a 37°C, 5% CO2 incubator. After 4 hours, the culture medium was replaced with fresh RPMI 1640 supplemented with 10% fetal bovine serum and cultured for an additional 48 hours before subsequent testing.

[0179] (4) Construction of bone marrow-derived macrophages overexpressing Angulin-1

[0180] Concentrated viral particles, pLVX-Angulin-1, were added to a culture of primary bone marrow macrophages from wild-type C57BL / 6J mice at an MOI of 10. 8 μg / mL polybrene was also added, gently mixed, and incubated in a 37°C, 5% CO2 incubator. After 4 hours, the culture medium was replaced with fresh RPMI 1640 supplemented with 10% fetal bovine serum and cultured for an additional 48 hours before subsequent testing.

[0181] (5) Angulin-1 overexpression virus pLVX-Angulin-1 was constructed using the following method:

[0182] ① Construction of plasmid

[0183] a) Obtain target gene: The coding sequence of mouse gene Angulin-1 was obtained by PCR amplification from mouse cDNA library. The coding sequences of mouse gene Angulin-1 are

[0184] SEQ ID NO.1: https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001164184.1;

[0185] SEQ ID NO.2: https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001164185.1;

[0186] SEQ ID NO.3: https: / / www.ncbi.nlm.nih.gov / nuccore / NM_017405.2.

[0187] Specific primers were designed, an EcoRI restriction site was added to the 5' end of the upstream primer, and a SmaI restriction site was added to the 5' end of the downstream primer. The primer sequences were verified to be correct before synthesis.

[0188] b) Vector Treatment: Double digest the pLVX-Puro vector with EcoRI and SmaI. The digestion system is as follows: 1 μg of pLVX vector, 1 μL of EcoRI, 1 μL of SmaI, 2 μL of 10× Buffer, and ddH2O to 20 μL. Incubate at 37°C for 2 h. After digestion, perform agarose gel electrophoresis to recover the linearized vector fragment.

[0189] c) Ligation reaction: Mix the recovered target gene fragment with the linearized pLVX-Puro vector at a molar ratio of 3:1. Add 1 μL of T4 DNA ligase and 2 μL of 10× T4 DNA ligase buffer, and make up to 20 μL with ddH2O. Incubate the mixture at 16°C overnight.

[0190] d) Transformation and Screening: Transform the ligation product into competent E. coli DH5α cells, spread onto solid LB medium containing ampicillin, and incubate at 37°C for 12-16 hours. Pick a single colony and inoculate it into liquid LB medium containing ampicillin. Incubate at 37°C with shaking overnight. Isolate the plasmid, perform double enzyme digestion, and verify by sequencing to identify the correct recombinant plasmid, pLVX-Angulin-1.

[0191] ② 293T cell culture

[0192] 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. When the cell density reached 80%-90%, they were subcultured. The cells were digested with 0.25% trypsin and passaged at a 1:3 ratio.

[0193] ③ Virus packaging and concentration

[0194] a) Co-transfection of 293T cells: 293T cells in logarithmic growth phase were seeded into 6-well plates. Transfection was performed when the cell density reached 70%-80%. Using lipofectamine transfection, the recombinant plasmid pLVX-Angulin-1 was mixed with the packaging plasmids psPAX2 and pMD2.G at a ratio of 4:3:1. Lipofectamine reagent was added and the procedure was performed according to the reagent manufacturer's instructions. Six hours after transfection, the medium was replaced with fresh DMEM.

[0195] b) Virus supernatant collection: 48 h and 72 h after transfection, collect the cell culture supernatant and filter through a 0.45 μm filter to remove cell debris.

[0196] c) Virus concentration: Transfer the filtered viral supernatant to an ultracentrifuge tube and ultracentrifuge at 25,000 rpm at 4°C for 2 h. Discard the supernatant and resuspend the viral pellet in an appropriate amount of PBS to obtain a concentrated viral solution.

[0197] ④ Infect primary peritoneal macrophages or bone marrow-derived macrophages

[0198] 2. ox-LDL uptake experiment

[0199] Same as Example 1.

[0200] 3. Foam cell detection: Macrophage Oil Red O staining

[0201] Same as Example 1.

[0202] 4. Data Analysis

[0203] Prism 8 software was used to analyze the experimental data. Unpaired t-test was used and the data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

[0204] 5. Experimental results

[0205] The results showed that overexpression of Angulin-1 in wild-type mouse bone marrow-derived macrophages and peritoneal macrophages could inhibit the uptake of dil-ox-LDL (e.g. Figure 24 and Figure 25 ) and ox-LDL-mediated foam cell formation (as shown Figure 26 and Figure 27 shown).

[0206] Example 6

[0207] Restoration of Angulin-1 expression levels in bone marrow transplantation experiments inhibits Angulin-1 MKO LDLR - / - Study of the development of atherosclerosis in mice.

[0208] 1. Experimental Animals

[0209] (1) LDLR - / - mice

[0210] Same as Example 3

[0211] (2) Bone marrow transplantation

[0212] ① Extraction of bone marrow cells from donor mice: Same as Example 4.

[0213] ② Preparation of bone marrow transplant recipient mice: recipient mice are Angulin-1 MKO LDLR - / - The operation was the same as in Example 4 to form chimera Angulin-1 MKO →Angulin-1 MKO LDLR - / - Mice and Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - mouse.

[0214] 2. Modeling method

[0215] Angulin-1 MKO →Angulin-1 MKO LDLR - / - Mice and Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - Mice were fed a high cholesterol diet (containing 1.25% cholesterol and 20% fat) and euthanized after 30 weeks. Peritoneal macrophages and aorta were collected for subsequent testing (e.g. Figure 28 shown).

[0216] 3. Extraction of peritoneal macrophages

[0217] Same as Example 1.

[0218] 4. Plaque area and stability detection

[0219] Same as Example 2.

[0220] 5. Data Analysis

[0221] Prism 8 software was used to analyze the experimental data. Unpaired t-test was used and the data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

[0222] 6. Experimental Results

[0223] Angulin-1 MKO →Angulin-1 MKO LDLR - / - Compared with mice, Angulin-1 Ctrl →Angulin-1 MKO LDLR - / - The progression of atherosclerotic lesions in the entire aorta and aortic root of mice was significantly slowed down (eg Figure 29 and Figure 30 The proportion of foam cells in peritoneal macrophages was significantly reduced (as shown in Figure 31 As shown in Figure 2), it was demonstrated that the restoration of Angulin-1 expression in macrophages could alleviate the MKO LDLR - / - Development of atherosclerosis in mice.

[0224] The present study found that macrophage Angulin-1 deficiency leads to LDLR - / - The plaque area and necrotic core area of ​​mice increased significantly, promoting the occurrence and development of atherosclerosis; the bone marrow transplantation model revealed that the deficiency of Angulin-1 in macrophages increased LDLR in patients with a high cholesterol diet. - / - The formation and development of atherosclerotic plaques in mice; and the LDLR - / - After the expression level of Angulin-1 in macrophages of mice returned to normal, the plaque area and necrotic core area of ​​their aorta were significantly reduced, effectively inhibiting the occurrence and development of atherosclerosis (such as Figure 32 shown).

[0225] It should be noted that, in this article, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0226] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments described herein, but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. Use of macrophage Angulin-1 gene and / or Angulin-1 protein in the preparation or screening of drugs for preventing, alleviating, adjuvant therapy, or / and treating atherosclerosis and related cardiovascular diseases.

2. Use of macrophage Angulin-1 gene and / or Angulin-1 protein promoters in the preparation of drugs for preventing, alleviating, assisting in the treatment and / or treatment of atherosclerosis and related cardiovascular diseases, and in enhancing the expression and / or activity and / or function of the Angulin-1 gene and / or Angulin-1 protein.

3. The use according to claim 2, characterized in that The promoter includes: a recombinant expression vector containing an Angulin-1 gene or a gene fragment having more than 90% homology with the Angulin-1 gene, and at least one of a polypeptide, protein, nucleic acid, nucleic acid aptamer, natural active substance, biological preparation, and compound capable of upregulating the expression of the Angulin-1 gene and / or Angulin-1 protein.

4. The use according to claim 2, characterized in that The recombinant expression vector includes any one of a vector-based eukaryotic expression plasmid, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, LNP liposome, microinjection technology, gene editing system elements or homologous recombination vector that specifically targets the Angulin-1 gene.

5. The use according to claim 2, characterized in that The expression promoter of the Angulin-1 gene and / or Angulin-1 protein targets and regulates the expression of the Angulin-1 gene and / or Angulin-1 protein by regulating the signal pathway.

6. A drug, characterized in that The drug comprises at least one of the Angulin-1 gene and / or Angulin-1 protein according to claim 1, or the expression promoter of the Angulin-1 gene and / or Angulin-1 protein according to any one of claims 2 to 5, and uses them as the main functional components of the drug.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients, which include at least one of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, and a lubricant. The drug is in the form of any of tablets, capsules, powders, pills, granules, solutions, suspensions, syrups, injections, suppositories, inhalants, and sprays.

8. The medicine according to claim 6, characterized in that The content of the functional component in the medicine is 1%-99%.

9. Use of a reagent for detecting the expression of Angulin-1 gene and / or protein in macrophages in the preparation of a product for auxiliary diagnosis, screening or evaluation of atherosclerosis and related cardiovascular diseases.

10. The use according to claim 9, characterized in that The product includes any one of a chip, a diagnostic reagent, and a diagnostic kit; the detection sample of the product is any one of cells, serum, and whole blood; the product contains an antibody that specifically binds to the Angulin-1 protein or a peptide segment of the Angulin-1 protein, or contains primers that specifically amplify the Angulin-1 gene, or contains a probe that specifically detects the Angulin-1 gene.

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