Application of macrophage SCAP as target spot in treatment or prevention of atherosclerosis

Abstract

The invention relates to the field of biological medicine, and discloses application of SCAP as a target spot in treatment or prevention of atherosclerosis. The invention proves that compared with normal control, SCAP expression of macrophages in aortic tissues of atherosclerosis patients is up-regulated. The invention proves that the progress of atherosclerosis can be obviously relieved by macrophage SCAP specific knockout. The invention also proves that macrophage SCAP specific knockout can improve aortic plaque lipid accumulation, and the local inflammation level is improved by influencing macrophage polarization to relieve atherosclerosis. According to the application, the application of the macrophage SCAP as a new target spot in treating or preventing the atherosclerosis is found.

Description

Technical Field

[0001] The present invention relates to the application of macrophage SCAP as a target in treating or preventing atherosclerosis, and belongs to the technical field of biomedicine. Background Art

[0002] Atherosclerotic cardiovascular disease (ASCVD) is one of the greatest threats to global public health, with a high incidence and a younger prevalence. In my country, the prevalence of coronary artery sclerosis ranges from approximately 4.3% to 8.8%. With the aging population, this figure is steadily increasing, severely impacting public health and quality of life. Atherosclerosis is considered the root cause of the vast majority of cardiovascular diseases. Therefore, in-depth research into the pathogenesis of atherosclerosis is crucial.

[0003] Macrophages are the core drivers of the pathological process of atherosclerosis, and are involved in the entire process from lipid deposition and inflammation initiation to plaque progression and rupture. In atherosclerosis (AS), macrophages are the core regulators throughout the entire process. When the vascular endothelium is damaged, monocytes are recruited to the intima by chemotactic factors and differentiate into macrophages. They take up oxidized low-density lipoprotein (ox-LDL) through scavenger receptors to form foam cells, initiating lipid deposition. And their polarization state determines the direction of the plaque: the pro-inflammatory M1 type secretes factors such as TNF-α, exacerbating inflammation, leading to the rupture of the fibrous cap, and ultimately triggering plaque rupture and thrombosis, leading to the occurrence of cardiovascular events.

[0004] SCAP, a chaperone protein, translocates SREBP from the endoplasmic reticulum to the Golgi apparatus, activating lipid metabolism and cholesterol synthesis. Dysregulation of the SCAP-SREBP pathway is closely associated with obesity, hypercholesterolemia, and increased risk of cardiovascular disease. As a confluence of inflammation and cholesterol metabolism, SCAP plays a crucial role in the development of atherosclerosis. VSMC-specific SCAP knockdown reduces lipid accumulation and intracellular oxidative stress, increases excess lipid clearance by enhancing autophagy through the ROS / AMPK pathway in VSMCs, inhibits VSMC foam cell formation, and alleviates atherosclerotic plaque formation, thereby alleviating atherosclerotic plaque formation. SCAP regulates VSMC NLRP3 inflammasome activation, thereby inducing endothelial dysfunction and enhancing atherosclerosis progression. SCAP plays a crucial role in atherosclerosis. Currently, there are no reports on the use of macrophage SCAP as a target for the treatment or prevention of atherosclerosis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new drug action target for clinical prevention and treatment of atherosclerosis.

[0006] In order to achieve the above-mentioned object, the present invention provides the use of macrophage SCAP as a target in the treatment and prevention of atherosclerosis.

[0007] The present invention demonstrates that SCAP expression is upregulated in atherosclerosis, and also demonstrates in animal experiments that macrophage SCAP-specific knockout can alleviate the development of atherosclerosis.

[0008] Specific knockout of macrophage SCAP reduces the area of ​​atherosclerotic plaques in mice, and at the same time can improve lipid accumulation in plaques of atherosclerotic mice, reduce macrophage infiltration, and by affecting macrophage polarization, reduce the local inflammation level of the plaque, alleviate the occurrence of atherosclerosis, and can be used for the prevention and treatment of cardiovascular diseases such as atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Two different genotypes of mice (APOE - / - Mouse model, namely SCAP △Mφ APOE - / - APOE mice and control group - / - Mouse model, namely SCAP fl / fl APOE - / - ) Schematic diagram of the animal modeling process.

[0010] Figure 2 In the figure, A is the expression of SCAP in all cells in single-cell sequencing of clinically asymptomatic and symptomatic human carotid artery samples in the GSE260657 dataset; B is the expression of SCAP in macrophages in single-cell sequencing of clinically asymptomatic and symptomatic human carotid artery samples in the GSE260657 dataset; C is the expression of SCAP in macrophages fed with NCD and PD diet for 12 weeks. fl / fl ;APOE - / - Fluorescence co-localization analysis of F4 / 80 and SCAP in rat aortic plaque sections; D is SCAP in rats fed with PD diet for 12 weeks fl / fl ;APOE - / - Immunohistochemical staining of SCAP in mouse aortic plaque sections.

[0011] Figure 3 Figure A shows SCAP fed with PD fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - The results of Oil Red O staining of the entire aorta of mice; Figure B is the quantitative statistical graph of Oil Red O detection of the entire aorta; Figure C is the SCAP of PD-fed fl / fl ;APOE - / - and SCAP △Mφ;APOE - / - Oil red O staining of the aortic sinus of mice; Panel D is the quantitative statistical graph of Oil red O detection in the aortic sinus.

[0012] Figure 4 Figure A in the middle shows SCAP fed with PD fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - HE staining of plaques in the aortic sinus of mice; B is a quantitative statistical graph of HE staining of plaques in the aortic sinus; C is Sirius red staining of plaques in the aortic sinus; D is a PD-fed SCAP fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - Quantitative statistical graph of Sirius red staining of plaques in the aortic sinus of mice.

[0013] Figure 5 Figure A shows SCAP fed with PD fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - F4 / 80 immunohistochemical staining of plaques in the aortic sinus of mice; Panel B is a quantitative statistical graph of F4 / 80 immunohistochemical staining of plaques in the aortic sinus; Panel C is a PD-fed SCAP fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - IL-8 immunohistochemical staining of plaques in the aortic sinus of mice; D is a quantitative statistical graph of IL-8 immunohistochemical staining of plaques in the aortic sinus of mice. E is a PD-fed SCAP fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - The mRNA expression levels of IL-1β, IL-6, and TNF-α in the aortic root of mice.

[0014] Figure 6 A in the middle is SCAP fed with PD fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - Fluorescence co-localization analysis of CD80 and F4 / 80 in the plaques of mouse aortic sinus. B is PD-fed SCAP fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / -Fluorescence co-localization analysis of CD206 and F4 / 80 in the plaques of mouse aortic sinus. C is SCAP fed with PD. fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - Immunohistochemical staining of CD80 in the plaques in the aortic sinus of mice. D is a quantitative statistical graph of CD80 immunohistochemical staining in the plaques in the aortic sinus of mice. E is a PD-fed SCAP. fl / fl ;APOE - / - and SCAP △Mφ ;APOE - / - Immunohistochemical staining of CD206 in the plaques in the aortic sinus of mice. Panel F is a quantitative statistical graph of the positive immunohistochemical staining of CD206 in the plaques in the aortic sinus of mice.

[0015] Among them: *, p < 0.05; **, p < 0.01; ***, p < 0.001. DETAILED DESCRIPTION

[0016] Below in conjunction with the accompanying drawings in the specific embodiments of the present invention, the technical scheme in the embodiments of the present invention is further fully explained, and the described embodiment is only a part of the embodiment in the present invention. After reading the content of the present invention, researchers in this field can understand the present invention more comprehensively. The experimental methods used in the present invention are all conventional methods in this area, and the reagents used are all commercially available. For the development of animal experiments, the experimental protocols are approved by the Animal Ethics Committee of Chongqing Medical University and follow the "Guide to the Care and Use of Laboratory Animals".

[0017] The mice used in this part of the experiment were 6-8 weeks old APOE- / - mice, SCAP flox mice (SCAP fl / fl), and SCAP △Mφ ;APOE - / - Mice (LysM-Cre mice and SCAP fl / fl Mice were mated to obtain macrophage SCAP-specific knockout mice, and then AopE - / - Mate and eventually obtain SCAP △Mφ ;APOE - / - Double transgenic mice), and SCAPfl / fl; APOE- / - mice. They were divided into the following groups: (1) SCAP fl / fl ;APOE - / - Mice were fed NCD diet for 12 weeks, (2) SCAP fl / fl ;APOE - / - Mouse, (3) SCAP △Mφ ;APOE - / -Mice were fed with a PD (Paigen) diet (containing 20% ​​fat, 1.25% cholesterol, and 0.45% sodium cholate) for 12 weeks to establish an atherosclerosis model.

[0018] Aorta Isolation: Mice were anesthetized with sodium pentobarbital and sacrificed. The viscera were removed to expose the aorta. Starting from the branching point of the iliac arteries, the surrounding tissue was gradually dissected until the bifurcation of the heart and carotid arteries was reached. Excess tissue surrounding the aorta was carefully dissected under a stereomicroscope and cut open at the point where the aorta root connected to the heart. The heart tissue was blotted dry and embedded in cryosection embedding medium for subsequent frozen section preparation. The entire length of the aorta was placed in a 10 ml EP tube containing 4% paraformaldehyde fixative for subsequent Oil Red O staining.

[0019] Oil Red O staining: The entire aorta was removed from the fixative and washed with PBS to remove any residual paraformaldehyde. An incision was made along the medial aspect of the aortic arch, and the ascending branches were carefully opened to ensure complete exposure of the vascular intima. The aorta was then decolorized by soaking in 60% isopropanol for 10 minutes and then placed in Oil Red O staining solution for 2 to 3 hours. The Oil Red O solution was discarded and the aorta was rinsed with 60% isopropanol until plaques appeared red and the arterial wall turned white. The aorta was then rinsed several times with distilled water, dried with absorbent paper, and placed on a black surface for photography.

[0020] Oil Red O staining of frozen tissue sections: Prepare Oil Red O staining solution (Oil Red O stock solution: ddH2O = 3:2), filter, and set aside. Remove frozen tissue sections from -20°C and rewarm at room temperature for 30 minutes. Fix with 4% paraformaldehyde for 15 minutes. Wash three times with PBS for 5 minutes. Add 60% isopropanol and decolorize for 5 seconds. Rinse twice with ddH2O. Incubate with hematoxylin for 2 minutes to stain the nuclei, then bluing with tap water for 5 minutes. Allow slides to dry at room temperature and then mount with neutral gum.

[0021] Hematoxylin and eosin (HE) staining: Mouse aorta tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections. The sections were dewaxed and rehydrated. Stained with hematoxylin for 5-10 minutes, then rinsed with tap water to remove excess stain for approximately 10 minutes. Stained with eosin for 30 seconds. After staining, the sections were dehydrated with 80%, 95%, and 100% ethanol for 5 minutes each, followed by xylene I and xylene II for 10 minutes each. After dehydration and transparency, the sections were mounted with mounting media. Observation was performed using a tissue section scanner. Sirius red staining: Mouse aorta tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections. The sections were dewaxed and rehydrated, then stained with Sirius red for 20 minutes. After washing with ddH2O, the sections were stained for nuclei, stained with hematoxylin for 8 minutes, and rinsed with tap water for 10 minutes to restore the blue color. After staining, the sections were dehydrated and transparentized with gradient ethanol and xylene, and then mounted. Observation was performed using a tissue section scanner.

[0022] Immunohistochemical staining: Paraffin sections were dewaxed and hydrated, followed by antigen retrieval using citrate buffer and three washes of 5 minutes each in PBST. Sections were incubated with an endogenous peroxidase blocker for 10 minutes and then with antibodies overnight at 4°C. Sections were washed three times with 5 minutes each in PBST, and then incubated with a biotinylated secondary antibody at room temperature for 20 minutes. DAB chromogen working solution was prepared at a ratio of 1:1 DAB solvent to chromogen, and DAB was used for development. Nuclear staining was performed with hematoxylin. After staining, sections were dehydrated, mounted, and examined using a tissue scanner.

[0023] Immunofluorescence staining: The frozen sections were placed at room temperature for 20 minutes to rewarm; fixed with 4% paraformaldehyde for 15 minutes; washed with PBS for 5 minutes, washed three times; permeabilized with 0.3%-0.5% TritonX-100 for 15 minutes; washed with PBS for 5 minutes, washed three times; blocked with 10% goat serum; incubated with primary antibody at 4°C overnight for 30 minutes and placed in a dark box; washed with PBS for 5 minutes, washed three times; incubated with fluorescent secondary antibody at 374°C for 1 hour; washed with PBS for 5 minutes, washed three times; stained with DAPI for 6 minutes; washed with PBS for 5 minutes, washed three times; mounted with anti-fluorescence quencher and observed under a microscope.

[0024] Real-time fluorescence quantitative PCR (qRT-PCR): (1) Extraction of tissue RNA: Add an appropriate volume of tissue to a tissue homogenate tube, add 1 mL of RNA lysis buffer, and fully disrupt it with a biological sample homogenizer. Transfer the homogenate to an enzyme-free EP tube, add 200 μl of chloroform and shake it thoroughly. After standing for 5 minutes, centrifuge it at 12,000 rpm in a 4°C centrifuge for 15 minutes. After centrifugation, take the upper layer solution to another EP tube, add 500 μl of isopropanol, mix it thoroughly, and let it stand for 10 minutes. Centrifuge it at 12,000 rpm in a 4°C centrifuge for 10 minutes. Discard the supernatant, add 75% ethanol to it, and centrifuge it again for 5 minutes. Discard the supernatant. After drying, add an appropriate amount of DEPC water and detect the concentration. (2) Reverse transcription: Prepare samples according to the conventional reverse transcription system within the research group and perform reverse transcription according to the conventional reaction procedure. (3) Real-time fluorescence quantitative PCR: The reaction procedure was as follows: 50°C, 2 min; 95°C, 5 min; 37°C, 20 s; 55°C, 2 s; 72°C, 30 s; for a total of 40 cycles.

[0025] Example 1 is a schematic diagram of the construction of the mouse animal model

[0026] Example 2 SCAP expression is upregulated during the progression of atherosclerosis

[0027] In the GSE260657 dataset, SCAP expression was upregulated in single-cell sequencing of clinically symptomatic carotid plaque samples compared with the human asymptomatic carotid plaque group ( Figure 2 A), SCAP expression is upregulated in macrophages ( Figure 2 B). Analysis of co-localization of F4 / 80 and SCAP by immunofluorescence staining ( Figure 2 C) Immunohistochemical staining of SCAP in mice fed with PD diet for 12 weeks fl / fl ;APOE - / - The expression of SCAP in aortic plaques was found to be upregulated ( Figure 2 D).

[0028] Example 3 Macrophage-specific SCAP knockdown slows lipid accumulation

[0029] The results of macroscopic oil red O staining showed that the aortic plaque area of ​​mice with macrophage SCAP specific knockdown was significantly reduced ( Figure 3 A, B); Oil red O staining of the aortic root showed that the aortic lipid deposition in mice with macrophage SCAP specific knockdown was significantly lower than that in the control group ( Figure 3 C, D)

[0030] Example 4 Macrophage-specific SCAP knockdown reduces plaque area

[0031] With SCAP fl / fl;APOE - / - Compared with the mice group, the aortic plaque area and necrotic core area of ​​the mice with macrophage SCAP knockdown were significantly reduced ( Figure 4 A, C).

[0032] Example 5 Macrophage-specific SCAP knockdown reduces local macrophage infiltration and inflammatory cytokine expression in the aorta

[0033] With SCAP fl / fl ;APOE - / - Compared with the mice group, macrophage SCAP-specific knockdown reduced macrophage infiltration ( Figure 5 A), reduced the level of local inflammation in the aorta ( Figure 5 B). The expression of inflammatory factors (IL-1β, IL-6 and TNFα) was decreased ( Figure 5 C).

[0034] Example 6 Macrophage-specific SCAP knockdown reduces macrophage M1 polarization and increases macrophage M2 polarization and SCAP fl / fl ;APOE - / - Compared with the mice group, macrophage SCAP-specific knockdown reduced macrophage M1 polarization ( Figure 6 A, C) increased macrophage M2 polarization ( Figure 6 B, E).

Claims

1. Application of macrophage SCAP as a target in the treatment or prevention of atherosclerosis.

2. The use according to claim 1, characterized in that Macrophage-specific knockout of SCAP ameliorates the progression of atherosclerosis.

3. The use according to claim 2, characterized in that Macrophage-specific knockout of SCAP can ameliorate lipid accumulation in aortic plaques.

4. The use according to claim 2, characterized in that Macrophage-specific knockout of SCAP ameliorates inflammatory responses in aortic plaques.

5. The use according to claim 1, characterized in that Macrophage-specific knockout of SCAP reduces plaque area in the aorta of atherosclerotic mice.

6. The use according to claim 4, characterized in that Macrophage SCAP-specific knockout can improve the inflammatory response in aortic plaques and the progression of atherosclerosis by affecting macrophage polarization.

Images