Application of SCAP as target spot in treatment or prevention of vascular smooth muscle cell senescence
By determining the role of SCAP in cholesterol-induced VSMC aging, a treatment or prevention method for SCAP as a target was proposed, which solved the problem of VSMC aging accelerated atherosclerosis and achieved the reduction of cellular aging and inflammation indicators.
Patent Information
- Application Number
- CN202411413282.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2025-04-01
AI Technical Summary
The prior art has not yet effectively solved the problem of aging of vascular smooth muscle cells (VSMC) accelerated the occurrence and development of atherosclerosis, and lacked the treatment or prevention means targeting SCAP.
By determining the role of SCAP in cholesterol-induced VSMC aging, it was proved that SCAP-specific overexpression was related to the upregulation of cellular senescence and inflammation indicators, and then proposed the treatment or prevention method of SCAP as a target.
In vitro experiments, the cellular senescence and inflammation indicators of VSMC-specific SCAP knockdown were significantly reduced, indicating that SCAP has potential effects as a target treatment or prevention method.
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Abstract
Description
Technical Field
[0001] The present invention relates to the application of SCAP as a target in treating or preventing senescence of vascular smooth muscle cells, and belongs to the technical field of biomedicine. Background Art
[0002] Atherosclerosis (AS) is the leading cause of cardiovascular disease (CVD), the leading cause of death worldwide. Cells within atherosclerotic plaques show signs of senescence, especially vascular smooth muscle cells (VSMCs), which originate from the medial layer of VSMCs. In addition, differentiated VSMCs are beneficial and necessary for plaque stability because they are the main source of collagen in the fibrous cap (FC) responsible for its tensile strength. VSMCs in human atherosclerotic plaques show progressive senescence and increased inflammation, which aggravates plaque instability and accelerates AS. According to literature reports, cholesterol can regulate cell growth and proliferation signaling pathways in a derivative-dependent (steroid hormones, bile acids) and derivative-independent manner, indicating that cholesterol metabolism disorders are related to a variety of aging-related diseases, but the mechanism by which cholesterol metabolism disorders cause aging is still unclear.
[0003] Sterol regulatory element binding protein (SREBP) cleavage activating protein (SCAP) is a cholesterol sensor that regulates signal transduction for intracellular cholesterol homeostasis. When sterols in cells are depleted, SCAP binds tightly to SREBP to form a complex that is transported from the endoplasmic reticulum to the Golgi apparatus, causing SREBPs to be cleaved and transported to the nucleus. SCAP dysregulation can affect the development of metabolic diseases. Studies have found that knocking down SCAP in VSMCs can significantly thin the vascular wall of the mouse aorta and lead to embryonic lethality, indicating that SCAP plays an important role in the cardiovascular system, but the role of SCAP in the development of atherosclerosis accelerated by aging of vascular smooth muscle cells has not been well studied.
[0004] Currently, there are no reports on the application of SCAP as a target in the treatment or prevention of VSMC aging. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a new drug action target for clinically preventing and treating accelerated atherosclerosis after VSMC aging.
[0006] To achieve the above object, the present invention provides the use of SCAP as a target in treating or preventing cholesterol-induced VSMC senescence.
[0007] The present invention firstly determines the relationship between VSMC aging and atherosclerosis, and demonstrates the up-regulated expression of β-galactosidase staining in atherosclerotic plaques in animal experiments, and demonstrates in vitro experiments that in cholesterol-induced VSMC aging, the cellular aging index of VSMC-specific SCAP overexpression is upregulated, while the cellular aging index of VSMC-specific SCAP knockdown is reduced.
[0008] The present invention determines that SCAP regulates aging by upregulating VSMC inflammatory indicators induced by cholesterol, thereby promoting the occurrence and development of atherosclerosis. In vitro experiments have shown that in cholesterol-induced VSMC aging, VSMC-specific overexpressed cell inflammatory indicators are upregulated, while VSMS-specific SCAP knockdown cell inflammatory indicators are downregulated. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 A is the HE staining analysis of aortic plaque sections of APOE knockout mice fed with PD diet and NCD diet for 12 weeks; B is the β-galactosidase staining analysis of aortic plaque sections of APOE knockout mice fed with PD diet and NCD diet for 12 weeks; C is the immunohistochemical analysis of SCAP in aortic plaque sections of APOE knockout mice fed with PD diet for 12 weeks; D is the fluorescence co-localization analysis of α-SMA and SCAP in aortic plaque sections of APOE knockout mice fed with PD diet for 12 weeks.
[0010] Figure 2 In the figure, A shows the expression of aging-related indicators detected by Western Blot after vascular smooth muscle cells were incubated with cholesterol for 24, 48, and 72 hours; B shows the β-galactosidase staining analysis of the experimental group and the control group after vascular smooth muscle cells were incubated with cholesterol for 72 hours.
[0011] Figure 3 In the figure, A shows the expression of inflammation-related indicators detected by Western Blot after vascular smooth muscle cells were incubated with cholesterol for 24, 48, and 72 hours; B shows the statistical chart of protein expression levels.
[0012] Figure 4 In the figure, A shows the results of Western Blot detection of SCAP after VSMCs overexpressing SCAP were incubated with cholesterol for 72 hours; B shows the expression of aging-related indicators after VSMCs overexpressing SCAP were incubated with cholesterol for 72 hours; C shows the expression of inflammation-related indicators after VSMCs overexpressing SCAP were incubated with cholesterol for 72 hours.
[0013] Figure 5In the figure, A is the result of Western Blot detection of SCAP after VSMC with SCAP knockdown was incubated with cholesterol for 72 hours; B is the expression of aging-related indicators by Western Blot after VSMC with SCAP knockdown was incubated with cholesterol for 72 hours; C is the expression of inflammation-related indicators by Western Blot after VSMC with SCAP knockdown was incubated with cholesterol for 72 hours. DETAILED DESCRIPTION
[0014] The present invention is further described below in conjunction with specific implementation methods to ensure that relevant personnel can better understand and implement the present invention, but the present invention is not limited thereby.
[0015] The materials and methods in the examples are as follows:
[0016] Animal Models and Treatments:
[0017] Six-week-old APOE- / - male mice purchased from Nanjing Model Animal Center were selected as animal experimental subjects, and they were fed with a PD diet (containing additional 40% fat + 1.25% cholesterol + 0.5% bile acid) for 12 weeks to successfully construct an AS mouse model.
[0018] Mouse model grouping: 6-week-old APOE- / - male mice were selected, and the experimental group was fed with a PD diet for 12 weeks; the control group was fed with an NCD diet for 12 weeks. The mice were directly killed and the aorta and heart were removed for dissection.
[0019] Hematoxylin and eosin (H&E) staining: Mouse carotid arteries were fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, cut into 5-μm sections, dewaxed, and stained with hematoxylin and eosin. After dehydration, differentiation, and sealing, a slice scanner was used for image acquisition and analysis.
[0020] Immunofluorescence staining: After dewaxing, the paraffin sections of the tissues were subjected to microwave antigen repair with citrate buffer. After blocking, antibodies to SCAP and α-SMA were added dropwise and placed at 4°C overnight. After washing with phosphate buffered saline (PBS), the sections were incubated with secondary antibodies and counterstained with DAPI. After dehydration with alcohol gradients and clearing with xylene, the sections were sealed with anti-fluorescence quenching sealing agents and photographed using a confocal microscope.
[0021] Cell culture and cell treatment: Mouse vascular smooth muscle cells (VSMCs) were obtained from the American Type Culture Collection (ATCC, VA, USA). VSMCs were cultured using basic DMEM\F12 medium (Gibco, Thermo Fisher Scientific, Eugene, Oregon, USA) supplemented with 10% fetal bovine serum (Natocor, Argentina) and 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Eugene, Oregon, USA). The cells were cultured in a cell culture incubator at 37°C in a humidified atmosphere of 5% CO2.
[0022] Western Blot: Use RIPA lysis buffer, add 1mM phosphatase inhibitor and 1mM protein phosphatase inhibitor, extract protein, add protein loading buffer, place in 100℃ metal bath for 10 minutes, perform SDS PAGE electrophoresis, and then transfer to membrane (250mA). After BSA blocking, add primary antibody, SCAP (1:3000) and β-ACTIN (1:5000), shake at 4℃ overnight. After washing, incubate with secondary antibody, and then use luminometer to expose.
[0023] β-Galactosidase staining: A β-galactosidase staining kit (Beyotime, C0602) was used for senescence staining. Briefly, VSMCs were fixed in 4% paraformaldehyde solution for 15 min, washed three times with PBS, incubated in a 37° oven overnight, and photographed under a microscope.
[0024] Immunohistochemical staining: After paraffin sections were dewaxed and dehydrated, antigen retrieval was performed using citrate solution, incubated with endogenous peroxidase blocker for 10 minutes, blocked with 10% goat serum for about 1 hour, and then incubated with SCAP antibody at 4° overnight. Biotin-labeled secondary antibody was incubated for 1 hour, horseradish peroxidase-labeled streptomycin working solution was incubated for 15 minutes, and then DAB color development was performed, followed by dehydration, transparency, sealing, and scanning.
[0025] Example 1 Upregulation of SCAP expression in atherosclerotic mice
[0026] APOE knockout mice were fed PD and NCD diets for 12 weeks and the sections of aortic plaques were analyzed by HE staining ( Figure 1 A); β-galactosidase staining was used to detect cell senescence in aortic plaques of APOE knockout mice fed with PD diet for 12 weeks ( Figure 1 B); Immunohistochemical staining was used to detect the expression of SCAP in aortic plaques of APOE knockout mice fed with PD diet for 12 weeks, and it was found that the expression of SCAP was upregulated ( Figure 1 C); Immunofluorescence staining was then used to analyze the co-localization of α-SMA and SCAP ( Figure 1 D).
[0027] Example 2 Role of SCAP in Cholesterol-Induced Smooth Muscle Cell Senescence
[0028] VSMCs were incubated with cholesterol for 24 hours, 48 hours, and 72 hours, and it was found that the senescence indicators of VSMCs were significantly upregulated 72 hours after cholesterol induction. Figure 2 A); Cholesterol was used to incubate VSMC for 72 hours, and β-galactosidase staining was significantly increased ( Figure 2 B).
[0029] Example 3 The relationship between SCAP and cholesterol-induced smooth muscle cell inflammatory response
[0030] VSMCs were incubated with cholesterol for 24 hours, 48 hours, and 72 hours, and it was found that the inflammatory indicators of VSMCs were significantly upregulated 72 hours after cholesterol induction. Figure 3 A); Statistical analysis of related proteins ( Figure 3 B).
[0031] Example 4: VSMC-specific SCAP overexpression aggravates the senescent phenotype and inflammatory response of smooth muscle cells
[0032] After incubating VSMCs overexpressing SCAP with cholesterol for 72 hours, Western Blot detected the upregulation of SCAP expression ( Figure 4 A); After VSMCs overexpressing SCAP were incubated with cholesterol for 72 hours, the related indicators of senescence phenotype were significantly upregulated ( Figure 4 B); After VSMCs overexpressing SCAP were incubated with cholesterol for 72 hours, inflammation-related indicators were significantly upregulated ( Figure 4 C).
[0033] Example 5 VSMC-specific SCAP knockdown alleviates the senescent phenotype and inflammatory response of smooth muscle cells
[0034] After incubating SCAP knockdown VSMC with cholesterol for 72 hours, Western Blot detected the downregulation of SCAP expression ( Figure 5 A); After 72 hours of cholesterol incubation of VSMCs with SCAP knockdown, the related indicators of aging phenotype were significantly downregulated ( Figure 5 B); After 72 hours of cholesterol incubation with SCAP-knockdown VSMCs, inflammation-related indicators were significantly knocked down ( Figure 5 C).
Claims
1. Application of SCAP as a target in the treatment or prevention of atherosclerosis.
2. The use according to claim 1, characterized in that The application of SCAP in cholesterol-induced VSMC senescence.
3. The use according to claim 2, characterized in that The SCAP is used to accelerate the occurrence and development of atherosclerosis in cholesterol-induced VSMC inflammatory response and cell senescence.
4. The use according to claim 1, comprising an in vitro application, wherein the aging and inflammatory indicators in VSMC cells that specifically overexpress SCAP are significantly increased compared with the control group; and the aging and inflammatory indicators in VSMC cells that specifically knock down SCAP are significantly reduced compared with the control group.